Expression and export of angiogenesis inhibitors as immunofusins

ABSTRACT

Disclosed are nucleotide sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-angiogenesis inhibitor fusion protein. The angiogenesis inhibitors can be angiostatin, endostatin, a plasminogen fragment having angiostatin activity, or a collagen XVIII fragment having endostatin activity. The nucleotide sequences can be inserted into a suitable expression vector and expressed in mammalian cells. Also disclosed is a family of immunoglobulin Fc-angiogenesis inhibitor fusion proteins that can be produced by expression of such nucleotide sequences. Also disclosed are methods using such nucleotide sequences and fusion proteins for treating conditions mediated by angiogenesis.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/097,883, filed on Aug. 25, 1998, which is incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to methods and compositions formaking and using fusion proteins containing an angiogenesis inhibitor.More particularly, the invention relates to methods and compositions formaking and using fusion proteins called immunofusins which contain animmunoglobulin Fc region and an angiogenesis inhibitor.

BACKGROUND OF THE INVENTION

[0003] Two potent angiogenesis inhibitors, angiostatin (O'Reilly et al.(1994) Cell 79:315) and endostatin (O'Reilly et al. (1997) Cell 88:277),were discovered and found to be generated naturally by primary tumors.Both proteins are specific inhibitors of endothelial cell proliferationand inhibit tumor growth by blocking angiogenesis, the formation of newblood vessels that nourish tumors. Studies have shown that theseangiogenesis inhibitors are non-toxic even at very high doses and thatthey may suppressed the growth of metastases and primary tumors mayregress to a dormant microscopic state. Both inhibitors were identifiedas proteolytic fragments of much larger intact molecules. Angiostatinwas found to be a fragment of plasminogen, and endostatin a fragment ofcollagen XVIII.

[0004] These two proteins have generated great interest in the cancerarea because they have been shown to suppress the growth of manydifferent types of tumors in mice, with no obvious side effects or drugresistance. Traditional chemotherapy generally leads to acquired drugresistance caused primarily by the genetic instability of cancer cells.Rather than targeting cancer cells, therapies using angiogenesisinhibitors target the normal endothelial cells, which support the growthof the tumor. Because endothelial cells are genetically stable, it ispossible that angiogenesis inhibitor therapies may result in less drugresistance. Studies indicate that drug resistance did not develop inmice exposed to prolonged anti-angiogenic therapy using endostatin.Furthermore, repeated cycles of endostatin treatment in mice resulted inprolonged tumor dormancy and no recurrence of tumors followingdiscontinuation of therapy (Boehm et al. (1997) Nature 390:404).

[0005] Despite promising results in mice, it has not been possible toproduce clinical grade soluble, active angiostatin and endostatin incommercial quantities using E. coli, baculoviral, yeast, and mammalianexpression systems. Expression in E. coli yielded insoluble proteinaggregates of undefined composition, which could not be injected intohumans. Other production methods, such as baculovirus and mammalianexpression systems, yielded very low levels of the recombinant proteins(O'Reilly et al. (1997) Cell 88:277).

[0006] The poor yields of the expression systems to date may beexplained by both angiostatin and endostatin being internal fragments ofmuch larger proteins. The truncated proteins may not fold properly inthe absence of the residues that are cleaved from the precursormolecules. For example, angiostatin has 26 cysteine residues which formnumerous disulfide bonds. Expression of angiostatin by itself may notprovide the optimal environment for these numerous disulfide bonds toform correctly in the secretory pathway. Also, the recombinantendostatin protein produced in E. coli precipitated during dialysis,possibly due to the hydrophobicity of endostatin (O'Reilly et al. (1997)Cell 88:277).

[0007] A major hurdle with the use of angiostatin and endostatin intheir present forms is that relatively large amounts of proteins have tobe injected daily for weeks to months to achieve the desired clinicaloutcome. For example, in current mouse models, dosages of 20 mg/kg/dayof endostatin are needed to demonstrate optimal efficacy (Boehm et al.(1997) Nature 390:404). Given that there is an urgent need to testendostatin and angiostatin clinically, a production method that cangenerate large quantities of clinical grade material is important.

[0008] One expression system that has been used to produce high levelexpression of fusion proteins in mammalian cells is a DNA constructencoding, a signal sequence, an immunoglobulin Fc region and a targetprotein. The fusion product of this construct generally is termed an“immunofusin.” Several target proteins have been expressed successfullyas immunofusins which include: IL2, CD26, Tat, Rev, OSF-2, βIG-H3, IgEReceptor, PSMA, and gp120. These expression constructs are disclosed inU.S. Pat. No. 5,541,087 and U.S. Pat. No. 5,726,044, the disclosures ofwhich are incorporated herein by reference.

[0009] A major purpose of expressing recombinant fusion proteins inmammalian cells has been to attempt to confer novel or useful propertiesto the hybrid molecules, e.g., proper folding, increased solubility,targeting of a cytokine or toxin in vivo, Fe receptor binding,complement fixation, protein A binding, increased circulation half-life,and increased ability to cross the blood-brain barrier. Examples ofrecombinant fusion proteins produced in mammalian cells include cytokineimmunoconjugates (Gillies et al. (1992) Proc. Natl. Acad. Sci. USA89:1428; Gillies et al. (1993) Bioconjugate Chemistry 4:230),immunoadhesins (Capon et al. (1989) Nature 337:525), immunotoxins(Chaudhary et al. (1989) Nature 339:394), and a nerve growth factorconjugate (Friden et al. (1993) Science 259:373). Each of the foregoingpublications is incorporated herein by reference.

[0010] It is an object of the invention to provide novel DNAs whichfacilitate efficient production and secretion of angiogenesis inhibitorsin a variety of mammalian host cells. It is another object of theinvention to provide methods for treating mammals with nucleic acidsencoding, or amino acid sequences defining angiogenesis inhibitorproteins, including non-native, biosynthetic, or otherwise artificialproteins such as proteins which have been created by rational design.

SUMMARY OF THE INVENTION

[0011] The present invention features methods and compositions useful inmaking and using fusion proteins containing an angiogenesis inhibitorprotein. The fusion proteins can facilitate a high level expression ofbiologically active angiogenesis inhibitor proteins. The angiogenesisinhibitor proteins can then be cleaved from the fusion protein andcombined with a pharmaceutically acceptable carrier prior toadministration to a mammal, for example, a human. Alternatively, nucleicsequences encoding, or amino acid sequences defining the angiogenesisinhibitor containing fusion proteins can be combined with apharmaceutically acceptable carrier and administered to the mammal.

[0012] In one aspect, the invention provides nucleic acid molecules, forexample, DNA or RNA molecules, encoding a fusion protein of theinvention. The nucleic acid molecule encodes a signal sequence, animmunoglobulin Fc region, and at least one target protein, also referredto herein as the angiogenesis inhibitor protein, selected from the groupconsisting of angiostatin, endostatin, a plasminogen fragment havingangiostatin activity, a collagen XVIII fragment having endostatinactivity, and combinations thereof. In a preferred embodiment, thenucleic acid molecule encodes, serially in a 5′ to 3′ direction, thesignal sequence, the immunoglobulin Fc region and the target proteinsequence. In another preferred embodiment, the nucleic acid moleculeencodes, serially in a 5′ to 3′ direction, the signal sequence, thetarget sequence, and immunoglobulin Fc region.

[0013] In another preferred embodiment, the immunoglobulin Fc regioncomprises an immunoglobulin hinge region and preferably comprises atleast one immunoglobulin constant heavy region, for example, animmunoglobulin constant heavy 2 (CH₂) domain, an immunoglobulin constantheavy 3 (CH₃) domain), and depending upon the type of immunoglobulinused to generate the Fc region, optionally an immunoglobulin constantheavy region 4 (CH4) domain. In a more preferred embodiment, theimmunoglobulin Fc region comprises a hinge region, a CH₂ domain and aCH₃ domain. Under certain circumstances, the immunoglobulin Fc regionpreferably lacks at least the CH₁, domain. Although the immunoglobulinFc regions may be based on any immunoglobulin class, for example, IgA,IgD, IgE, IgG, and IgM, immunoglobulin Fc regions based on IgG arepreferred.

[0014] In another embodiment, the nucleic acid of the invention can beincorporated in operative association into a replicable expressionvector which can then be transfected into a mammalian host cell. Inanother preferred embodiment, the invention provides host cellsharboring such nucleic acid sequences of the invention.

[0015] In another aspect, the invention provides a fusion proteincomprising an immunoglobulin Fc region linked, either directly through apolypeptide bond or by means of a polypeptide linker, to a targetprotein selected from the group consisting of angiostatin, endostatin, aplasminogen fragment having angiostatin activity, a collagen XVIIIfragment having endostatin activity, and combinations thereof. Thetarget protein may be fused via its C-terminal end to an N-terminal endof the immunoglobulin Fc region. However, in a more preferred embodimentthe target protein is fused via its N-terminal end to a C-terminal endof the immunoglobulin Fc region.

[0016] In another embodiment, the fusion protein may comprise a secondtarget protein selected from the group consisting of angiostatin,endostatin, a plasminogen fragment having angiostatin activity, and acollagen XVIII fragment having endostatin activity. In this type ofconstruct the first and second target proteins can be the same ordifferent proteins. For example, in a preferred embodiment, the fusionprotein comprises a first target protein of angiostatin, animmunoglobulin Fc region and a second target protein of endostatin. Thefirst and second target proteins may be linked together, either directlyor by means of a polypeptide linker. Alternatively, both target proteinsmay be linked, either directly or via a polypeptide linker, to theimmunoglobulin Fc region. In the latter case, the first target proteinis connected to an N-terminal end of the immunoglobulin Fc region andthe second target protein is connected to a C-terminal end of theimmunoglobulin Fc region.

[0017] In another embodiment, two fusion proteins may associate, eithercovalently, for example, by a disulfide or peptide bond, ornon-covalently, to produce a multimeric protein. In a preferredembodiment, two fusion proteins are associated covalently by means ofone or more disulfide bonds through cysteine residues, preferablylocated within immunoglobulin hinge regions disposed within theimmunoglobulin Fc regions of both chains.

[0018] In a preferred embodiment, the target protein comprises aplasminogen fragment having a molecular weight of approximately 40 kDand, optionally comprises, an amino acid sequence as set forth in SEQ IDNO: 3. In another preferred embodiment, the target protein comprises acollagen XVIII fragment having an amino acid sequence set forth in SEQID NO: 1. Furthermore, the target protein can be full-length angiostatinor endostatin or bioactive fragments thereof. The source of the targetprotein in generating certain fusion proteins will depend upon theintended use of the target protein. For example, if the target proteinis to be administered to a human, the target protein preferably is ofhuman origin.

[0019] In another aspect, the invention provides methods of producing afusion protein comprising an immunoglobulin Fc region and a targetprotein selected from the group consisting of angiostatin, endostatin, aplasminogen fragment having angiostatin activity, and a collagen XVIIIfragment having endostatin activity. The method comprises the steps of(a) providing a mammalian cell containing a DNA molecule encoding such afusion protein, either with or without a signal sequence, and (b)culturing the mammalian cell to produce the fusion protein. Theresulting fusion protein can then be harvested, refolded, if necessary,and purified using conventional purification techniques well known andused in the art. Assuming that the fusion protein comprises aproteolytic cleavage site disposed between the immunoglobulin Fc regionand the target protein, the target can be cleaved from the fusionprotein using conventional proteolytic enzymes and if necessary,purified prior to use.

[0020] In another aspect, the invention provides methods for treatingmammals, for example, a human, in need of an angiogenesis inhibitorbased therapy. For example, it is contemplated that the angiogenesisinhibitors of the invention may be administered to a human afflictedwith a tumor. Treatment with the angiogenesis inhibitor may slow down orstop tumor growth and, under certain circumstances, may cause tumorregression. Treatment may include administering to the mammal an amountof the angiogenesis inhibitor in an amount sufficient to slow down orstop tumor growth. The angiogenesis inhibitor may be provided in theform of a fusion protein or as a nucleic acid, preferably operativelyassociated with an expression vector, in combination with apharmaceutically acceptable carrier.

[0021] The foregoing and other objects, features and advantages of thepresent invention will be made more apparent from the detaileddescription, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1A-1F are schematic illustrations of exemplary angiogenesisinhibitor fusion proteins constructed in accordance with the invention(see Examples 10-15). The Figures depict, respectively, FIG. 1A,Fc-Kringle 1 of Angiostatin; FIG. 1B, Fc-inner Kringle 1 of Angiostatin;FIG. 1C, Fc-Endostatin-GlySer linker-inner Kringle 1 of Angiostatin;FIG. 1D, Fc-Endostatin-GlySer linker-Kringle 1 of Angiostatin; FIG. 1E,Fc-Endostatin-GlySer linker-Angiostatin; FIG. 1F,Angiostatin-Fc-Endostatin. The vertical lines represent optionaldisulfide bonds connecting cysteine residues (C) disposal within a hingeregion of the Fc molecule.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention provides fusion proteins, referred to herein asimmunofusins, which were useful in the production of commercialquantities of clinical grade angiogenesis inhibitors. The angiogenesisinhibitors may be cleaved from the immunofusin protein constructs priorto use. However, it is contemplated that the immunofusins or nucleicacids encoding the immunofusins may be administered directly to mammalsin need of treatment with an angiogenesis inhibitor.

[0024] The invention thus provides fusion proteins comprising animmunoglobulin Fc region and at least one target protein, referred toherein as an angiogenesis inhibitor. The angiogenesis inhibitorpreferably is selected from the group consisting of angiostatin,endostatin, a plasminogen fragment angiostatin activity, a collagenXVIII fragment having endostatin activity. It is contemplated, however,that other polypeptides having angiogenesis inhibitor activity, nowknown or late discovered, may be expressed as fusion proteins of thetype described herein.

[0025] Six exemplary embodiments of protein constructs embodying theinvention are illustrated in the drawing as FIGS. 1A-1F. Because dimericconstructs are preferred, all are illustrated as dimers cross-linked bya pair of disulfide bonds between cysteines on adjacent subunits. In thedrawings, the disulfide bridges are depicted as linking together theportions of two immunoglobulin Fc regions via an immunoglobulin hingeregion, and thus are characteristic of native forms of these molecules.While constructs including the hinge region of Fc are preferred and havebeen shown promise as therapeutic agents, the invention contemplatesthat the crosslinking at other positions may be chosen as desired.Furthermore, under some circumstances, dimers or multimers useful in thepractice of the invention may be produced by non-covalent association,for example, by hydrophobic interaction.

[0026] Because homodimeric constructs are important embodiments of theinvention, FIG. 1 illustrates such constructs. It should be appreciatedthat heterodimeric structures also are useful but, as is known to thoseskilled in the art, often can be difficult to purify. However, viableconstructs useful to inhibit angiogenesis in various mammalian species,including humans, can be constructed comprising a mixture of homodimersand heterodimers. For example, one chain of the heterodimeric structuremay comprise endostatin and the another may comprise angiostatin.

[0027]FIG. 1A illustrates a dimer construct produced in accordance withthe procedure set forth in Example 10. Each monomer of the dimercomprises an immunoglobulin Fc region 1 including a hinge region, a CH₂domain and a CH₃ domain. Attached directly to the C terminus of the Fcregion 1 is the first Kringle region of angiostatin 2, both inner andouter rings. FIG. 1B shows a second embodiment of the invention (seeExample 11) comprising the same Fc region as in FIG. 1A, this timehaving only the inner ring of Kringle one of angiostatin 3 attached tothe C terminal end of the Fc region 1. FIGS. 1C through 1E depictvarious embodiments of the protein constructs of the invention, whichinclude as a target protein plural angiogenesis inhibitors arranged intandem and connected by a linker. In FIG. 1C, the target proteincomprises full-length endostatin 4, a polypeptide linker 5, and theinner ring of Kringle one of angiostatin 3. FIG. 1D depicts a proteincomprising an Fc region the same as that of FIG. 1A and a target proteincomprising a full-length endostatin 4, a polypeptide linker 5, and afull Kringle one region of angiostatin (both inner and outer rings) 2.FIG. 1E differs from the construct of FIG. 1D in that the most Cterminal protein domain comprises a full-length copy of angiostatin 7.

[0028] Although FIGS. 1A-1E represent Fc-X type constructs, where X isthe target protein, it is contemplated that X-Fc type constructs mayalso be useful in the practice of the invention. Furthermore, it iscontemplated the useful proteins of the invention may also be depictedby the formula X-Fc-X, wherein the Xs may represent the same ordifferent target proteins. FIG. 1F depicts such a construct whichcomprises in an N- to C-terminal direction, full-length humanangiostatin 7, a human immunoglobulin Fc region 6 including a hingeregion, and full-length human endostatin domain 4.

[0029] The term “angiogenesis inhibitor,” as used herein, refers to anypolypeptide chain that reduces or inhibits the formation of new bloodvessels in a mammal. With regard to cancer therapy, the angiogenesisinhibitor reduces or inhibits the formation of new blood vessels in oron a tumor, preferably in or on a solid tumor. It is contemplated thatuseful angiogenesis inhibitors may be identified using a variety ofassays well known and used in the art. Such assays include, for example,the bovine capillary endothelial cell proliferation assay, the chickchorioallantoic membrane (CAM) assay or the mouse corneal assay.However, the CAM assay is preferred (see, for example, O'Reilly et al.(1994) Cell 79: 315-328 and O'Reilly et al. (1997) Cell 88: 277-285, thedisclosures of which are incorporated herein by reference). Briefly,embryos with intact yolks are removed from fertilized three day oldwhite eggs and placed in a petri dish. After incubation at 37° C., 3%CO₂ for three days, a methylcellulose disk containing the putativeangiogenesis inhibitor is applied to the chorioallantoic membrane of anindividual embryo. After incubation for about 48 hours, thechorioallantoic membranes were observed under a microscope for evidenceof zones of inhibition.

[0030] Preferred angiogenesis inhibitors useful in the practice of theinvention include, for example, angiostatin (O'Reilly et al. (1994) Cell79: 315-328, and U.S. Pat. Nos. 5,733,876; 5,837,682; and 5,885,795),and endostatin (O'Reilly et al. (1997) Cell 88: 277-285 and U.S. Pat.No. 5,854,205). As stated previously, angiostatin and endostatin arespecific inhibitors of endothelial cell proliferation and are capable ofinhibiting tumor growth by blocking angiogenesis, the formation of newblood vessels that nourish tumors.

[0031] Angiostatin has been identified as a proteolytic fragment ofplasminogen (O'Reilly et al. (1994) Cell 79: 315-328, and U.S. Pat. Nos.5,733,876; 5,837,682; and 5,885,795, the disclosure of which isincorporated herein by reference). Specifically, angiostatin is a 38 kDainternal fragment of plasminogen containing at least three of theKringle regions of plasminogen. Endostatin has been identified as aproteolytic fragment of collagen XVIII (O'Reilly et al. (1997) Cell 88:277-285, the disclosure of which is incorporated herein by reference).Specifically, endostatin is a 20 kDa C-terminal fragment of collagenXVIII. The terms “angiostatin” and “endostatin,” as used herein, refernot only to the full length proteins, but also to variants and bioactivefragments thereof, as well as to bioactive fragments of plasminogen andcollagen XVIII, respectively. The term bioactive fragment, with respectto angiostatin refers to any protein fragment of plasminogen orangiostatin that has at least 30%, more preferably at least 70%, andmost preferably at least 90% of the activity of full-length angiostatinas determined by the CAM assay. The term bioactive fragment, withrespect to endostatin refers to any protein fragment of collagen XVIIIor endostatin that has at least 30%, more preferably at least 70% andmost preferably at least 90% of the activity of full length endostatinas determined by the CAM assay.

[0032] The term variants includes specifies and allelic variants, aswell as other naturally occurring or non-naturally occurring variants,for example, generated by conventional genetic engineering protocols,that are at least 70% similar or 60% identical, more preferably at least75% similar or 65% identical, and most preferably 80% similar or 70%identical to either the naturally-occurring sequences of endostatin orangiostatin disclosed herein.

[0033] To determine whether a candidate polypeptide has the requisitepercentage similarity or identity to a reference polypeptide, thecandidate amino acid sequence and the reference amino acid sequence arefirst aligned using the dynamic programming algorithm described in Smithand Waterman (1981), J. Mol. Biol. 147:195-197, in combination with theBLOSUM62 substitution matrix described in FIG. 2 of Henikoff andHenikoff (1992), “Amino acid substitution matrices from protein blocks”,Proc. Natl. Acad Sci. USA 89:10915-10919. For the present invention, anappropriate value for the gap insertion penalty is −12, and anappropriate value for the gap extension penalty is −4. Computer programsperforming alignments using the algorithm of Smith-Waterman and theBLOSUM62 matrix, such as the GCG program suite (Oxford Molecular Group,Oxford, England), are commercially available and widely used by thoseskilled in the art.

[0034] Once the alignment between the candidate and reference sequenceis made, a percent similarity score may be calculated. The individualamino acids of each sequence are compared sequentially according totheir similarity to each other. If the value in the BLOSUM62 matrixcorresponding to the two aligned amino acids is zero or a negativenumber, the pair-wise similarity score is zero; otherwise the pair-wisesimilarity score is 1.0. The raw similarity score is the sum of thepair-wise similarity scores of the aligned amino acids. The raw scorethen is normalized by dividing it by the number of amino acids in thesmaller of the candidate or reference sequences. The normalized rawscore is the percent similarity. Alternatively, to calculate a percentidentity, the aligned amino acids of each sequence again are comparedsequentially. If the amino acids are non-identical, the pair-wiseidentity score is zero; otherwise the pair-wise identity score is 1.0.The raw identity score is the sum of the identical aligned amino acids.The raw score is then normalized by dividing it by the number of aminoacids in the smaller of the candidate or reference sequences. Thenormalized raw score is the percent identity. Insertions and deletionsare ignored for the purposes of calculating percent similarity andidentity. Accordingly, gap penalties are not used in this calculation,although they are used in the initial alignment.

[0035] The target proteins disclosed herein are expressed as fusionproteins with an Fc region of an immunoglobulin. As is known, eachimmunoglobulin heavy chain constant region is comprised of four or fivedomains. The domains are named sequentially as follows:CH₁-hinge-CH₂-CH₃(-CH₄). The DNA sequences of the heavy chain domainshave cross-homology among the immunoglobulin classes, e.g., the CH₂domain of IgG is homologous to the CH₂ domain of IgA and IgD, and to theCH₃ domain of IgM and IgE.

[0036] As used herein, the term, “immunoglobulin Fc region” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH₁ domain, a CH₂ domain, and a CH₃ domain, 2) a CH₁,domain and a CH₂ domain, 3) a CH₁, domain and a CH₃ domain, 4) a CH₂domain and a CH₃ domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment the Fc regionused in the DNA construct includes at least an immunoglobulin hingeregion a CH₂ domain and a CH₃ domain and preferably lacks at least theCH₁, domain.

[0037] The currently preferred class of immunoglobulin from which theheavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2,3, or 4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE(Igε) and IgM (Igμ), may be used. The choice of appropriateimmunoglobulin heavy chain constant regions is discussed in detail inU.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particularimmunoglobulin heavy chain constant region sequences from certainimmunoglobulin classes and subclasses to achieve a particular result isconsidered to be within the level of skill in the art. The portion ofthe DNA construct encoding the immunoglobulin Fc region preferablycomprises at least a portion of a hinge domain, and preferably at leasta portion of a CH₃ domain of Fcγ or the homologous domains in any ofIgA, IgD, IgE, or IgM.

[0038] Depending on the application, constant region genes from speciesother than human e.g., mouse or rat may be used. The Fc region used as afusion partner in the immunofusin DNA construct generally may be fromany mammalian species. Where it is undesirable to elicit an immuneresponse in the host cell or animal against the Fc region, the Fc regionmay be derived from the same species as the host cell or animal. Forexample, human Fc can be used when the host animal or cell is human;likewise, murine Fc can be used where the host animal or cell will be amouse. Further, substitution or deletion of constructs of these constantregions, in which one or more amino acid residues of the constant regiondomains are substituted or deleted also would be useful. One examplewould be to introduce amino acid substitutions in the upper CH₂ regionto create a Fc variant with reduced affinity for Fc receptors (Cole etal. (1997) J. Immunol. 159:3613). One of ordinary skill in the art canprepare such constructs using well known molecular biology techniques.

[0039] The use of human Fcγ1 as the Fc region sequence has severaladvantages. For example, if the angiogenesis inhibitor Fc fusion proteinis to be used as a biopharmaceutical, the Fcγ1 domain may confer theeffector function activities to the fusion protein. The effectorfunction activities include the biological activities such as complementfixation, antibody-directed cellular cytotoxicity, placental transfer,and increased serum half-life. The Fc domain also provides for detectionby anti-Fc ELISA and purification through binding to Staphylococcusaureus protein A (“Protein A”). In certain applications, however, it maybe desirable to delete specific effector functions from the Fc region,such as Fc receptor binding or complement fixation.

[0040] In the case of angiogenesis inhibitor immunofusins, one functionof the immunoglobulin Fc fusion partner is to facilitate proper foldingof the angiogenesis inhibitor protein to yield active angiogenesisinhibitor protein and to impact solubility to the active moieties, atleast in the extracellular medium. Since the Fc fusion partner ishydrophilic, the angiogenesis inhibitor immunofusin readily is solubleunlike, for example, the recombinant endostatin produced in E. coli(O'Reilly (1997) Cell 88:277.)

[0041] In all of the Examples disclosed herein, high levels ofproduction of the immunofusins were obtained. The angiogenesis inhibitorimmunofusins were secreted into media at concentrations typically ofabout 30 to 100 μg/ml, and could be purified readily to homogeneity byProtein A chromatography. In addition, the angiogenesis inhibitorimmunofusins could be cleaved and further purified using conventionalpurification protocols using, for example, by heparin sepharose, lysinesepharose or affinity purification.

[0042] In addition to the high levels of expression, fusion proteins ofthe invention also exhibit longer serum half-lives, presumably due totheir larger molecular sizes. For example, human Fc-human angiostatinhas a serum half-life of 33 hours in mouse, as compared to 4-6 hours forhuman angiostatin (O'Reilly et al. (1996) Nature Medicine 2:689). It isbelieve that angiostatin with a molecular weight of 40 kD, andendostatin with a molecular weight of 20 kD, are small enough to becleared efficiently by renal filtration. In contrast, the dimeric formsof Fc-angiostatin and dimeric Fc-endostatin are 145 kD and 100 kD,respectively, because there are two immunoglobulin Fc regions attachedto either two angiostatin molecules or two endostatin molecules. Such abivalent structure may exhibit a higher binding affinity to theangiostatin or endostatin receptor. If the angiogenesis inhibitingactivity is receptor-mediated, the Fc fusion proteins are potentiallymore effective to suppress tumors than monovalent angiostatin ormonovalent endostatin by themselves. Furthermore, if angiostatin and/orendostatin belong to a class of dimeric protein ligands, the physicalconstraint imposed by the Fc on angiostatin or endostatin would make thedimerization an intramolecular process, thus shifting the equilibrium infavor of the dimer and enhancing its binding to the receptor. Cysteineresidues can also be introduced by standard recombinant DNA technologyto the monomer at appropriate places to stabilize the dimer throughcovalent disulfide bond formation.

[0043] As used herein, the term “multivalent” refers to a recombinantmolecule that incorporates two or more biologically active segments. Theprotein fragments forming the multivalent molecule may be linked througha polypeptide peptide linker which attaches the constituent partswithout causing a frame shift and permits each to functionindependently.

[0044] As used herein, the term “bivalent” refers to a multivalentrecombinant molecule having two target proteins in a fusion construct ofthe invention, e.g., an Fc-X molecule, where X independently is selectedfrom angiostatin, endostatin, or a variant thereof. Since there are twoX moieties fused to an immunoglobulin Fc region (which typically itselfis a dimer of the heavy chain fragments including at least a portion ofthe hinge region and CH₃ domain, and optionally the CH₂ domain), themolecule is bivalent (see, e.g., FIG. 1A). If the fusion construct ofthe invention has the form Fc-X-X, the resulting Fc dimer molecule istetravalent. The two proteins forming the Fc-X-X molecule may be linkedthrough a peptide linker. A bivalent molecule can increase the apparentbinding affinity between the molecule and its receptor. For instance, ifone endostatin moiety of an Fc-endostatin can bind to a receptor on acell with a certain affinity, the second endostatin moiety of the sameFc-endostatin may bind to a second receptor on the same cell with a muchhigher avidity (apparent affinity). This is because of the physicalproximity of the second endostatin moiety to the receptor after thefirst endostatin moiety is already bound. In the case of an antibodybinding to an antigen, the apparent affinity is increased by at least10⁴.

[0045] As used herein, the terms “multimer” and “multimeric” refers tothe stable association of two or more polypeptide chains eithercovalently, for example, by means of covalent interaction, for example,by a disulfide bond or non-covalently, for example, by hydrophobicinteraction. The term multimer is intended to encompass bothhomomultimers, wherein the polypeptides are the same, as well asheteromultimers, wherein the polypeptides are different.

[0046] As used herein, the term “dimeric” refers to a specificmultimeric molecule where two protein polypeptide chains are stablyassociated through covalent or non-covalent interactions. It should beunderstood that the immunoglobulin Fc region Fc fragment itselftypically is a dimer of the heavy chain fragments including at least aportion of the hinge region and CH₃ domain, and optionally the CH₂domain. Many protein ligands are known to bind to their receptors as adimer. If a protein ligand X dimerizes naturally, the X moiety in anFc-X molecule will dimerize to a much greater extent, since thedimerization process is concentration dependent. The physical proximityof the two X moieties connected by associated immunoglobulin Fc regionwould make the dimerization an intramolecular process, greatly shiftingthe equilibrium in favor of the dimer and enhancing its binding to thereceptor.

[0047] It is understood that the present invention exploits conventionalrecombinant DNA methodologies for generating the Fe fusion proteinsuseful in the practice of the invention. The Fc fusion constructspreferably are generated at the DNA level, and the resulting DNAsintegrated into expression vectors, and expressed to produce theimmunofusins. As used herein, the term “vector” is understood to meanany nucleic acid comprising a nucleotide sequence competent to beincorporated into a host cell and to be recombined with and integratedinto the host cell genome, or to replicate autonomously as an episome.Such vectors include linear nucleic acids, plasmids, phagemids, cosmids,RNA vectors, viral vectors and the like. Non-limiting examples of aviral vector include a retrovirus, an adenovirus and an adeno-associatedvirus. As used herein, the term “gene expression” or “expression” of atarget protein, is understood to mean the transcription of a DNAsequence, translation of the mRNA transcript, and secretion of an Fcfusion protein product.

[0048] A useful expression vector is pdCs (Lo et al. (1988) ProteinEngineering 11:495, the disclosure of which is incorporated herein byreference) in which the transcription of the Fc-X gene utilizes theenhancer/promoter of the human cytomegalovirus and the SV40polyadenylation signal. The enhancer and promoter sequence of the humancytomegalovirus used was derived from nucleotides −601 to +7 of thesequence provided in Boshart et al., 1985, Cell 41:521, the disclosureof which is incorporated herein by reference. The vector also containsthe mutant dihydrofolate reductase gene as a selection marker (Simonsenand Levinson (1983) Proc. Nat. Acad. Sci. USA 80:2495, the disclosure ofwhich is incorporated herein by reference).

[0049] An appropriate host cell can be transformed or transfected withthe DNA sequence of the invention, and utilized for the expression andsecretion of a target protein. Currently preferred host cells for use inthe invention include immortal hybridoma cells, NS/O myeloma cells, 293cells, Chinese hamster ovary cells, Hela cells, and COS cells.

[0050] The fusion proteins of the invention preferably are generated byconventional recombinant DNA methodologies. The fusion proteinspreferably are produced by expression in a host cell of a DNA moleculeencoding a signal sequence, an immunoglobulin Fc region and a targetprotein (also referred to herein as an angiogenesis inhibitor).Preferred constructs may encode in a 5′ to 3′ direction, the signalsequence, the immunoglobulin Fc region and the target protein.Alternatively, the constructs may encode in a 5′ to 3′ direction, thesignal sequence, the target protein and the immunoglobulin Fc region.

[0051] As used herein, the term “signal sequence” is understood to meana peptide segment which directs the secretion of the angiogenesisinhibitor immunofusin protein and is thereafter cleaved followingtranslation in the host cell. The signal sequence of the invention is apolynucleotide, which encodes an amino acid sequence that initiatestransport of a protein across the membrane of the endoplasmic reticulum.Signal sequences which will be useful in the invention include antibodylight chain signal sequences, e.g., antibody 14.18 (Gillies et. al.,1989, Jour. of Immunol. Meth., 125:191-202), antibody heavy chain signalsequences, e.g., the MOPC141 antibody heavy chain signal sequence(Sakano et al., 1980, Nature 286:5774), and any other signal sequenceswhich are known in the art (see for example, Watson, 1984, Nucleic AcidsResearch 12:5145). Each of these references is incorporated herein byreference.

[0052] Signal sequences have been well characterized in the art and areknown typically to contain 16 to 30 amino acid residues, and may containgreater or fewer amino acid residues. A typical signal peptide consistsof three regions: a basic N-terminal region, a central hydrophobicregion, and a more polar C-terminal region. The central hydrophobicregion contains 4 to 12 hydrophobic residues that anchor the signalpeptide across the membrane lipid bilayer during transport of thenascent polypeptide. Following initiation, the signal peptide is usuallycleaved within the lumen of the endoplasmic reticulum by cellularenzymes known as signal peptidases. Potential cleavage sites of thesignal peptide generally follow the “(−3, −1) rule.” Thus a typicalsignal peptide has small, neutral amino acid residues in positions −1and −3 and lacks proline residues in this region. The signal peptidasewill cleave such a signal peptide between the −1 and +1 amino acids.Thus, the portion of the DNA encoding the signal sequence may be cleavedfrom the amino-terminus of the immunofusin protein during secretion.This results in the secretion of a immunofusin protein consisting of theFc region and the target protein. A detailed discussion of signalpeptide sequences is provided by von Heijne (1986) Nucleic Acids Res.,14:4683 the disclosure of which is incorporated herein by reference.

[0053] As would be apparent to one of skill in the art, the suitabilityof a particular signal sequence for use in the invention may requiresome routine experimentation. Such experimentation will includedetermining the ability of the signal sequence to direct the secretionof an immunofusin and also a determination of the optimal configuration,genomic or cDNA, of the sequence to be used in order to achieveefficient secretion of immunofusins. Additionally, one skilled in theart is capable of creating a synthetic signal peptide following therules presented by von Heijne, referenced above, and testing for theefficacy of such a synthetic signal sequence by routine experimentation.A signal sequence may also be referred to as a “signal peptide,” “leadersequence,” or “leader peptide.”

[0054] The fusion of the signal sequence and the immunoglobulin Fcregion is sometimes referred to herein as secretion cassette. Anexemplary secretion cassette useful in the practice of the invention isa polynucleotide encoding, in a 5′ to 3′ direction, a signal sequence ofan immunoglobulin light chain gene and an Fcγ1 region of the humanimmunoglobulin γ1 gene. The Fcγ1 region of the immunoglobulin Fcγ1 genepreferably includes at least a portion of the hinge domain and at leasta portion of the CH₃ domain, or alternatively at least portions of thehinge domain, CH₂ domain and CH₃ domain. The DNA encoding the secretioncassette can be in its genomic configuration or its cDNA configuration.

[0055] In another embodiment, the DNA sequence encodes a proteolyticcleavage site interposed between the secretion cassette and theangiogenesis inhibitor protein. A cleavage site provides for theproteolytic cleavage of the encoded fusion protein thus separating theFc domain from the angiogenesis inhibitor protein. As used herein,“proteolytic cleavage site” is understood to mean amino acid sequenceswhich are preferentially cleaved by a proteolytic enzyme or otherproteolytic cleavage agents. Useful proteolytic cleavage sites includeamino acids sequences which are recognized by proteolytic enzymes suchas trypsin, plasmin or enterokinase K. Many cleavage site/cleavage agentpairs are known. See, for example, U.S. Pat. No. 5,726,044, thedisclosure of which is incorporated herein by reference. Where thetarget protein sequence is a precursor molecule to angiostatin,endostatin, or an active variant thereof, the desired protein productmay be produced by cleavage with the endogenous proteolytic enzyme, suchas elastin or plasmin or urokinase.

[0056] The present invention also encompasses fusion proteins containingdifferent combinations of recombinant angiostatin and endostatin, orfragments thereof, which can be made in large quantities. Despite thedemonstrated efficacy in suppressing tumor growth, the mechanism of howangiostatin and endostatin block angiogenesis is not completely known.Angiostatin has several Kringle structures and endostatin has differentstructural motifs, each of which may be solely responsible for or assistin binding of the proteins to endothelial cells and exerting ananti-angiogenic effect. Accordingly, this invention includes targetproteins which are bioactive fragments of angiostatin, such as Kringle1, Kringle 2, Kringle 3, and combinations thereof, and endostatin whichexhibit physiologically similar behavior to naturally occurringfull-length angiostatin and endostatin.

[0057] Another embodiment of the present invention provides forbifunctional hybrid constructs of angiogenesis inhibitors. As usedherein, a bifunctional hybrid molecule or construct means a proteinproduced by combining two protein subunits, where the two subunits canbe derived from different proteins. Each protein subunit has its ownindependent function so that in the hybrid molecule, the functions ofthe two subunits may be additive or synergistic. Such functional hybridproteins would allow the synergistic effect of angiostatin andendostatin to be explored in animal models. A preferred bifunctionalhybrid may comprise at least two different angiogenesis inhibitorslinked in tandem, either directly or by means of a polypeptide linker.For example, in a preferred embodiment, the target sequence encodes atleast a portion of angiostatin linked in frame with at least a portionof endostatin and both the angiostatin and endostatin domains exhibitanti angiogenesis activity or angiogenesis inhibition. The two units maybe linked by a polypeptide linker.

[0058] As used herein the term “polypeptide linker is understood to meanan peptide sequence that can link two proteins together or a protein andan Fc region. The polypeptide linker preferably comprises a plurality ofamino acids such as glycine and/or serine. Preferably, the polypeptidelinker comprises a series of glycine and serine peptides about 10-15residues in length. See, for example, U.S. Pat. No. 5,258,698, thedisclosure of which is incorporated herein by reference. It iscontemplated however, that the optimal linker length and amino acidcomposition may be determined by routine experimentation.

[0059] It is found that when different parts of the angiostatin areexpressed as Fc fusion molecules, high levels of expression areobtained, presumably because the Fc portion acts as a carrier, helpingthe polypeptide at the C-terminus to fold correctly. In addition, the Fcregion can be glycosylated and highly charged at physiological pH, thusthe Fc region can help to solubilize hydrophobic proteins.

[0060] The present invention also provides methods for the production ofangiostatin and endostatin of non-human species as Fc fusion proteins.Non-human angiogenesis inhibitor fusion proteins are useful forpreclinical studies of angiogenesis inhibitors because efficacy andtoxicity studies of a protein drug must be performed in animal modelsystems before testing in humans. A human protein may not work in amouse model because the protein may elicit an immune response, and/orexhibit different pharmacokinentics skewing the test results. Therefore,the equivalent mouse protein is the best surrogate for the human proteinfor testing in a mouse model.

[0061] The standard Lewis lung carcinoma model in mice (O'Reilly et al.(1997) Cell 88:277) was used to compare soluble huFc-huAngiostatin,huFc-huEndostatin, muFc-muAngiostatin, muFc-muEndostatin with theinsoluble proteins produced in an E. Coli expression system. The solubleFc fusion proteins were more efficacious in suppressing tumor growth inthe Lewis lung model than the corresponding proteins produced in E.coli. Furthermore, laboratory mice are inbred and their tumors areinduced and not spontaneous. Therefore, efficacy in a mouse model maynot correlate to probable efficacy against human tumors. Preclinicalstudies in dogs will provide more precise information about the efficacyof these angiogenesis inhibitors on spontaneous tumors because there arenumerous naturally occurring, spontaneous canine tumors. The methods ofproducing murine (mu) Fc-mu angiostatin, muFc-mu endostatin, and canine(ca) Fc-ca angiostatin, caFc-ca endostatin of the present invention willfacilitate preclinical studies of angiogenesis inhibitors in both murineand canine systems.

[0062] The present invention provides methods of treating a conditionmediated by angiogenesis by administering the DNA, RNA or proteins ofthe invention. Conditions mediated by angiogenesis include, for example:solid tumors; blood born tumors, tumor metastasis, benign tumorsincluding hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyrogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenicdiseases (diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma) retrolentalfibroplasia, rubeosis, Osler-Webber Syndrome; myocardial angiogenesis;plaque neovascularization; telangiectasia; hemophiliac joints'angiofibroma; and wound granulation; and excessive or abnormalstimulation of endothelial cells, intestinal adhesions,artherosclerosis, sclerodermal and hypertrophic scars, i.e., keloids.

[0063] The DNA constructs disclosed herein can be useful in gene therapyprocedures in which the endostatin or angiostatin gene is delivered intoa cell by one of various means e.g., native DNA associated with apromoter or DNA within a viral vector. Once inside a cell, theangiostatin and/or endostatin gene or gene fragment is expressed and theprotein is produced in vivo to carry out its normal biological function.The DNA construct of the present invention results in high levels ofexpression of the fusion protein. The fusion proteins of the presentinvention may also be useful in treating conditions mediated byangiogenesis and may have greater clinical efficacy than nativeangiogenesis inhibitors and other recombinant angiogenesis inhibitorsbecause the angiogenesis inhibitor immunofusins of the present inventionhave a longer serum half-life than the other recombinant angiogenesisinhibitors or native angiogenesis inhibitors alone. The bivalent anddimeric forms of the present invention should have higher bindingaffinity due to the bivalent and dimeric structure. The bifunctionalhybrid molecules of the present invention may have a higher clinicalefficacy due to possible synergistic effects of two differentangiogenesis inhibitors connected by the fused Fc region or a flexiblepolypeptide linker.

[0064] The compositions of the present invention may be provided to ananimal by any suitable means, directly (e.g., locally, as by injection,implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the composition is tobe provided parenterally, such as by intravenous, subcutaneous,ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal,intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracisternal, intracapsular, intranasalor by aerosol administration, the composition preferably comprises partof an aqueous or physiologically compatible fluid suspension orsolution. Thus, the carrier or vehicle is physiologically acceptable sothat in addition to delivery of the desired composition to the patient,it does not otherwise adversely affect the patient's electrolyte and/orvolume balance. The fluid medium for the agent thus can comprise normalphysiologic saline (e.g., 9.85% aqueous NaCl, 0.15 M, pH 7-7.4).

[0065] Preferred dosages of the immunofusins per administration arewithin the range of 50 ng/m² to 1 g/m², more preferably 5 μg/m² to 200mg/m², and most preferably 0.1 mg/m² to 50 mg/m². Preferred dosages ofnucleic acids encoding the immunofusins per administration are withinthe range of 1 μg/m² to 100 mg/m², more preferably 20 μg/m² to 10 mg/m²,and most preferably 400 μg/m² to 4 mg/m². It is contemplated, however,that the optimal modes of administration, and dosages may be determinedby routine experimentation well within the level of skill in the art.

[0066] The invention is illustrated further by the followingnon-limiting examples.

EXAMPLES Example 1 Expression of huFc-huEndostatin

[0067] Human endostatin was expressed as a human Fc-human endostatin(huFc-huEndo) fusion protein according to the teachings of Lo et al.(1998) Protein Engineering 11:495. Fc refers to the Fc fragment of thehuman immunoglobulin gamma (DNA sequence set forth in SEQ ID NO: 1;amino acid sequence set forth in SEQ ID NO: 2). (Polymerase chainreactions PCR) was used to adapt the endostatin cDNA (SEQ ID NO: 3;whose amino acid sequence is disclosed in SEQ ID NO: 4), for expressionin an Fc-Endo fusion protein. The forward primer was either 5′-CC CCGGGT AAA CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 5; encoded amino acidsdisclosed in SEQ ID NO: 6) or 5′-C AAG CTT CAC AGC CAC CGC GAC TTC C(SEQ ID NO: 7; encoded amino acids disclosed in SEQ ID NO: 8), where theXmaI site or the HindIII site was followed by sequence encoding theN-terminus of endostatin. The primer with the XmaI site adapted theendostatin cDNA for ligation to the XmaI site at the end of the CH₃domain of the IgGFc region. The primer with the HindIII site adapted theendostatin cDNA for ligation to the HindIII site of the pdCs-Fc(D₄K)vector, which contains the enterokinase recognition site Asp₄-Lys(LaVallie et al. (1993) J. Biol. Chem. 268:23311-23317) at the junctionof the fusion protein. The reverse primer was 5′-C CTC GAG CTA CTT GGAGGC AGT CAT G (SEQ ID NO: 9), which was designed to put a translationSTOP codon (anticodon, CTA) immediately after the C-terminus ofendostatin, and this was followed by an XhoI site. The PCR products werecloned and sequenced, and the XmaI-XhoI fragment was ligated to theresulting XmaI and XhoI digested pdCs-Fc vector. Similarly, theHindIII-XhoI fragment encoding endostatin was ligated into appropriatelydigested pdCs-huFc(D₄K) vector. Stable clones expressing Fc-endo orFc(D₄K)-endostatin were obtained by electroporation of NS/0 cellsfollowed by selection in growth medium containing 100 nM methotrexate.Protein expression level was assayed by anti-human Fc ELISA (Example 3)and confirmed by SDS-PAGE, which showed a protein product of ˜52 kD. Thebest producing clones were subcloned by limiting dilutions.

Example 2 Cell Culture and Transfection

[0068] For transient transfection, the plasmid was introduced into humankidney 293 cells by co-precipitation of plasmid DNA with calciumphosphate (Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual,Cold Spring Harbor, N.Y.) or by lipofection using LipofectAMINE Plus(Life Technologies, Gaithersburg, Md.) according to supplier's protocol.

[0069] In order to obtain stably transfected clones, plasmid DNA wasintroduced into the mouse myeloma NS/0 cells by electroporation. NS/0cells were grown in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum. About 5×10⁶ cells were washed once with PBS andresuspended in 0.5 ml PBS. Ten μg of linearized plasmid DNA then wasincubated with the cells in a Gene Pulser Cuvette (0.4 cm electrode gap,BioRad, Hercules, Calif.) on ice for 10 min. Electroporation wasperformed using a Gene Pulser (BioRad, Hercules, Calif.) with settingsat 0.25 V and 500 μF. Cells were allowed to recover for 10 min. on ice,after which they were resuspended in growth medium and then plated ontotwo 96 well plates. Stably transfected clones were selected by growth inthe presence of 100 nM methotrexate (MTX), which was introduced two dayspost-transfection. The cells were fed every 3 days for three more times,and MTX-resistant clones appeared in 2 to 3 weeks. Supernatants fromclones were assayed by anti-Fc ELISA to identify high producers. Highproducing clones were isolated and propagated in growth mediumcontaining 100 nM MTX.

Example 3 ELISA Procedures

[0070] Three different ELISAs were used to determine the concentrationsof protein products in the supernatants of MTX-resistant clones andother test samples. The anti-human Fc (huFc) ELISA was used to measurethe amount of human Fc-containing proteins. The anti-murine Fc (muFc)and anti-canine Fc (caFc) antibodies were used in ELISAs to measure theamount of murine Fc-and canineFc-containing proteins, respectively. Theprocedure for the anti-huFc ELISA is described in detail herein below.

[0071] A. Coating Plates

[0072] ELISA plates were coated with AffiniPure Goat anti-Human IgG(H+L) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at 5 μg/mlin PBS, and 100 μl/well in 96-well plates (Nunc-Immuno plate MaxiSorp™,Nalge Nunc International, Rochester, N.Y. ). Coated plates were coveredand incubated at 4° C. overnight. Plates then were washed 4 times with0.05% Tween 20 in PBS, and blocked with 1% BSA/1% Goat Serum in PBS, 200μl/well. After incubation with the blocking buffer at 37° C. for 2hours, the plates were washed 4 times with 0.05% Tween in PBS and tappeddry on paper towels.

[0073] B. Incubation with Test Samples and Secondary Antibody

[0074] Test samples were diluted to the proper concentrations in asample buffer, containing 1% BSA/1% Goat Serum/0.05% Tween in PBS. Astandard curve was prepared with a chimeric antibody (with a human Fc),the concentration of which was known. To prepare a standard curve,serial dilutions were made in the sample buffer to give a standard curveranging from 125 ng/ml to 3.9 ng/ml. The diluted samples and standardswere added to the plate, 100 μl/well and the plate was then incubated at37° C. for 2 hr. After incubation, the plate was washed 8 times with0.05% Tween in PBS. To each well was then added 100 μl of secondaryantibody, the horse radish peroxidase (HRP)-conjugated anti-human IgG(Jackson ImmunoResearch Laboratories, Inc. West Grove, Pa), dilutedabout 1:120,000 in sample buffer. The exact dilution of the secondaryantibody had to be determined for each lot of the HRP-conjugatedAnti-Human IgG. After incubation at 37° C. for 2 hr, the plate waswashed 8 times with 0.05% Tween in PBS.

[0075] C. Development

[0076] A substrate solution was prepared by dissolving 30 mg (1 tablet)of o-phenylenediamine dihydrochloride (OPD) into 15 ml of 0.025 M citricacid/0.05 M Na₂HPO₄ buffer, pH 5, containing 0.03% of freshly addedH₂O₂. The substrate solution was added to the plate at 100 μl/well. Thecolor was allowed to develop for 30 min. at room temperature in thedark. The developing time can be subject to change, depending on lot tolot variability of the coated plates, the secondary antibody, etc. Thereaction was stopped by adding 4N H₂SO₄, 100 μ/well. The plate was readby a plate reader, which was set at both 490 and 650 nm, and programmedto subtract the background OD at 650 nm from the OD at 490 nm.

[0077] The procedure for the anti-muFc ELISA was similar, except thatELISA plate was coated with AffiniPure Goat anti-murine IgG (H+L)(Jackson ImmunoResearch, West Grove, Pa.) at 5 μg/ml in PBS, and 100μl/well; and the secondary antibody was horse radishperoxidase-conjugated goat anti-muIgG, Fcγ (Jackson ImmunoResearch WestGrove, Pa.), used at 1 in 5000 dilution. Similarly, for the anti-caFcELISA, the ELISA plate was coated with AffiniPure Rabbit anti-dog IgG,Fe Fragment specific (Jackson ImmunoResearch, West Grove, Pa.) at 5μg/ml in PBS, and 100 μl/well; and the secondary antibody was horseradish peroxidase-conjugated AffiniPure rabbit anti-dog IgG, Fc fragmentspecific (Jackson ImmunoResearch, West Grove, Pa.), used at 1 in 5000dilution.

Example 4 Expression of huFc-huAngiostatin

[0078] Human angiostatin (DNA sequence set forth in SEQ ID NO: 10; aminoacid sequence set forth in SEQ ID NO: 11) was expressed as a humanFc-human angiostatin (huFc-huAngio) fusion protein essentially asdescribed in Example 1. PCR was used to adapt the angiostatin cDNA (SEQID NO: 3), for expression in the pdCs-huFc or pdCs-huFc(D₄K) vectors.The respective forward primers were 5′-CC CCG GGT AAG AAA GTG TAT CTCTCA GAG (SEQ ID NO 12; encoded amino acids disclosed in SEQ ID NO: 13),and 5′-C CCC AAG CTT AAA GTG TAT CTC TCA GAG (SEQ ID NO: 14; encodedamino acids disclosed in SEQ ID NO: 15), where the XmaI site or theHindIII site was followed by sequence encoding the N-terminus ofangiostatin. The reverse primer was 5′-CCC CTC GAG CTA CGC TTC TGT TCCTGA GCA (SEQ ID NO: 16), which was designed to put a translation STOPcodon (anticodon, CTA) immediately after the C-terminus of angiostatin,and this was followed by an XhoI site. The PCR products were cloned andsequenced, and the resulting XmaI-XhoI fragment and the HindIII-XhoIfragment encoding angiostatin were ligated to the pdCs-huFc and thepdCs-huFc(D₄K) vectors, respectively. Stable NS/0 clones expressinghuFc-huAngio and huFc(D₄K)-huAngio were selected and assayed asdescribed in Examples 2 and 3.

Example 5 Expression of muFc-mu-Endostatin

[0079] Murine endostatin (DNA sequence set forth in SEQ ID NO: 17; aminoacid sequence set forth in SEQ ID NO: 18) and murine Fe (DNA sequenceset forth in SEQ ID NO: 19; encoded amino acids set forth in SEQ ID NO:20) were expressed as a murine F-murine endostatin (muFc-muEndo) fusionprotein essentially as described in Example 1. PCR was used to adapt theendostatin cDNA (SEQ ID NO: 4), for expression in the pdCs-muFc(D₄K)vector. The forward primer was 5′-C CCC AAG CTT CAT ACT CAT CAG GAC TTTC (SEQ ID NO: 21; encoded amino acids disclosed in SEQ ID NO: 22), wherethe HindIII site was followed by sequence encoding the N-terminus ofendostatin. The reverse primer was 5′-CCC CTC GAG CTA TTT GGA GAA AGAGGT C (SEQ ID NO: 23), which was designed to put a translation STOPcodon (anticodon, CTA) immediately after the C-terminus of endostatin,and this was followed by an XhoI site. The PCR product was cloned andsequenced, and the resulting HindIII-XhoI fragment encoding endostatinwas ligated into the pdCs-muFc(D₄K) vector. Stable NS/0 clonesexpressing muFc(D₄K)-muEndo were selected and assayed (anti-muFc ELISA)as described in Examples 2 and 3.

Example 6 Expression of muFc-muAngiostatin

[0080] Murine angiostatin (DNA sequence set forth in-SEQ ID NO: 24;amino acid sequence set forth in SEQ ID NO: 25) was expressed as amurine Fc-murine angiostatin (muFc-muAngio) fusion protein essentiallyas described in Example 1. PCR was used to adapt the angiostatin cDNA(SEQ ID NO: 6) for expression in the pdCs-Fc(D₄K) vector. The forwardprimer was 5′-C CCC AAG CTT GTG TAT CTG TCA GAA TGT AAG CCC TCC TGT CTCTGA GCA (SEQ ID NO: 26; encoded amino acids disclosed in SEQ ID NO: 27),where the HindIII site was followed by sequence encoding the N-terminusof angiostatin. The reverse primer was 5′-CCC CTC GAG CTA CCC TCC TGTCTC TGA GCA (SEQ ID NO: 28), which was designed to put a translationSTOP codon (anticodon, CTA) immediately after the C-terminus ofangiostatin, and this was followed by an XhoI site (CTCGAG). The PCRproduct was cloned and sequenced, and the HindIII-XhoI fragment encodingangiostatin was ligated to the pdCs-muFc(D₄K) vector. Stable NS/0 clonesexpressing muFc(D₄K)-muAngio were selected and assayed (anti-muFc ELISA)as described in Examples 2 and 3.

Example 7 Expression of Canine Fc (caFc)

[0081] Canine peripheral blood monocytic cells (PBMCs) isolated fromdog's blood were used to prepare mRNA. After synthesis of the firststrand cDNA with reverse transcriptase and oligo(dT), PCR was performedto amplify the canine Fc (Kazuhiko et al., (1992) JP 1992040894-A1)using the forward primer 5′-CC TTA AGC GAA AAT GGA AGA GTT CCT CGC (SEQID NO: 29; encoded amino acids disclosed in SEQ ID NO: 30), in which anAfIII site was introduced immediately upstream of the sequence encodingthe hinge region of the canine Fc, and the reverse primer 5′-C CTC GAGTCA TTT ACC CGG GGA ATG GGA GAG GGA TTT CTG (SEQ ID NO: 31), in which anXhoI site was introduced after the translation STOP codon (anticodon,TCA) of the canine Fc. The reverse primer also introduced a silentmutation to create a XmaI restriction site, which facilitates theconstruction of the pdCs-caFc(D₄K) vector through a linker-adaptor andligation to DNA constructs encoding canine endostatin or angiostatin.Similar to the construction of pdCs-huFc, which was described in detailin Lo et al. (Lo et al., Protein Engineering (1998) 11:495), theexpression vector for the pdCs-caFc was constructed as follows. TheAfIII-XhoI fragment encoding the canine Fc was ligated to the XbaI-AfIIIfragment encoding the light chain signal peptide and the XbaI-XhoIdigested pdCs vector. The resulting pdCs-caFc expression vector then wasused to transfect 293 cells. About 3 days post-transfection, thesupernatant was purified by Protein A chromatography. Expression of dogFc (DNA sequence set forth in SEQ ID NO: 32; amino acid sequence setforth in SEQ ID NO: 33) was confirmed by SDS-PAGE followed by Westernblot analysis using a peroxidase-conjugated Rabbit anti-Dog IgG, Fcfragment specific (Jackson ImmunoResearch, West Grove, Pa.).

Example 8 Expression of caFc-caEndostatin

[0082] The coding sequence for canine endostatin (DNA sequence set forthin SEQ ID NO: 34; amino acid sequence set forth in SEQ ID NO: 35) wasadapted to a HindIII-XhoI fragment for expression as a Fc fusionprotein, essentially as described in Example 5. At the 3′ end, a STOPcodon was introduced, for example, by PCR, immediately after the codonencoding the C-terminal lysine residue, and this was followed by theNotI restriction site. At the 5′ end, however, there was a DraIIIrestriction site convenient for reconstruction. An oligonucleotideduplex consisting of a HindIII and a DraIII sticky ends was chemicallysynthesized and used to ligate to the DraIII-XhoI restriction fragmentwhich encodes the rest of the canine endostatin cDNA. The duplex used isshown below:

[0083] HindIII

[0084] 5′-AGCTT CAC ACC CAC CAG GAC TTC CAG CCG GTG CTG CAC CTG (SEQ IDNO: 36)

[0085] A GTG TGG GTG GTC CTG AAG GTC GGC CAC GAC GTG-5′ (SEQ ID NO: 38)DraIII

[0086] The first CAC in the duplex encodes the N-terminal histidineresidue of the canine endostatin. The HindIII-XhoI fragment encoding thefull-length canine endostatin thus could be ligated to the HindIII-XhoIdigested pdCs-caFc vector (see Example 7) for expression. Stable NS/0clones expressing caFc-caEndo were selected and assayed by anti-caFcELISA, as described in Examples 2 and 3. The protein product wasanalyzed on SDS-PAGE and confirmed by Western blot analysis.

Example 9 Expression of caFc-caAngiostatin

[0087] The cDNA encoding the full length canine angiostatin (DNAsequence set forth in SEQ ID NO: 39; amino acid sequence set forth inSEQ ID NO: 40) was adapted for expression as a caFc fusion proteinessentially as in the aforementioned examples. Briefly, at the 3′ end, aSTOP codon was introduced, for example, by PCR, immediately after thecodon encoding the C-terminal lysine residue and this was followed by aNotI restriction site instead of an XhoI site, since there was aninternal XhoI restriction site in the cDNA of the canine angiostatin. Atthe 5′ end, a HindIII site was introduced in-frame immediately upstreamof the N-terminus of angiostatin. The HindIII-NotI fragment encoding thefull length canine angiostatin then was ligated to the HindIII-NotIdigested pdCs-caFc vector (where the NotI site was introduced at theXhoI site through linker ligation) for expression. Stable NS/0 clonesexpressing caFc-caAngio were selected and assayed by anti-caFc ELISA, asdescribed in Examples 2 and 3. The protein product was analyzed onSDS-PAGE and confirmed by Western blot analysis.

Example 10 Expression of muFc-K1 of muAngio

[0088] Angiostatin comprises the first four of the five Kringle domainsof plasminogen. To determine if any one or several Kringle domains areresponsible for the observed anti-angiogenic activity of angiostatin, itis possible to produce single Kringle domains by themselves orcombination thereof for testing. To demonstrate the utility of Fc as afusion protein partner, the expression of the first Kringle domain ofmurine angiostatin (K1) was achieved in the following way. The firstKringle domain ends at Glu-87 of murine angiostatin (SEQ ID NO: 25).There was a convenient NsiI restriction site in the cDNA at thisposition so that after digestion by NsiI, the four-base 3′-overhang wasremoved by T4 polymerase to create a blunt end. A translation STOP codonwas introduced immediately downstream of the GAA encoding Glu-87 vialigation to the palindromic linker TGA CTC GAG TCA (SEQ ID NO: 41),where the STOP codon TGA was followed by an XhoI site. The HindIII-XhoIfragment encoding this truncated angiostatin, i.e., first Kringle only,then was ligated into the pdCs-muFc(D₄K) vector for expression. Highlevels of expression were obtained in both transient and stableexpression, as analyzed by anti-muFc ELISA and SDS-PAGE.

Example 11 Expression of muFc-innerK1 of muAngio

[0089] A Kringle domain consists of multiple loops, including an outerloop and an inner loop. In the first Kringle of murine angiostatin, theinner loop is defined by Cys 55 and Cys 79, which together form adisulfide bond at the base of the loop. The Cys-67 of the inner loopforms another disulfide bond with a Cys residue of the outer loop togive the Kringle structure. To test if the inner loop has anyanti-angiogenic activity, it was expressed as a muFc-inner K1(Kringle 1) as follows. With a DNA fragment encoding the first Kringleas template, a mutagenic primer having the sequence 5′GGG CCT TGG AGCTAC ACT ACA (SEQ ID NO: 42; encoded amino acids disclosed in SEQ ID NO:43) was used to mutagenize TGC (Cys-67) to AGC (Ser), by PCR. Thisensures that the Cys-67 does not form a disulfide bond when the innerloop of Kringle 1 is expressed without the outer loop. An upstreamprimer having the sequence 5′GCGGATCCAAGCTT AGT ACA CAT CCC AAT GAG GG(SEQ ID NO: 44; encoded amino acids disclosed in SEQ ID NO: 45) was usedto introduce a HindIII site in frame immediately 5′ to the codon forSer-43 (AGT). A BamHI site was also introduced immediately upstream ofthe HindIII site. The BamHI site is useful for ligating to the BamHIsite at the end of the flexible Gly-Ser linker shown in Example 12below. Thus a HindIII-XhoI DNA fragment encoding Ser-43 through Glu-87of murine angiostatin was ligated to the pdCs-muFc(D₄K) vector forexpression. High levels of expression of muFc-innerK1 were obtained inboth transient and stable expression, as analyzed by anti-muFc ELISA andSDS-PAGE.

Example 12 Expression of muFc-muEndo-GlySer Linker-InnerK1 of muAngio

[0090] The hybrid molecule muFc-muEndo-innerK1 comprises muFc-muEndojoined by a polypeptide linker containing glycine and serine residues,to the inner loop of the first Kringle of murine angiostatin. The DNAconstruct was assembled as follows.

[0091] There is a BspHI site at the 3′ end of the murine endostatincDNA. To introduce a flexible linker of glycine and serine residues atthe C-terminus of murine endostatin, a 540-bp HindIII-BspHI fragmentencoding endostatin was ligated to an overlapping oligonucleotide duplexformed by the oligonucleotides disclosed in SEQ ID NO: 46 and SEQ ID NO:48. The amino acid linker encoded by SEQ ID NO: 46 is disclosed in SEQID NO: 47.

[0092] The HindIII-BamHI fragment encoding murine endostatin and theGly-Ser linker w a s subcloned into a standard cloning vector. The BamHIsite was then used to introduce the BamHI-XhoI fragment encoding theinnerK1 in Example 11. The resulting HindIII-XhoI fragment encodingmuEndo- GlySer linker-innerK1, was ligated to the pdCs-muFc(D₄K) vectorfor expression. High levels of expression of muFc-muEndo-GlySerlinker-innerK1 were obtained in both transient and stable expression, asanalyzed by anti-muFc ELISA and SDS-PAGE.

Example 13 Expression of muFc-muEndo-GlySer Linker-K1 of muAngio

[0093] The hybrid molecule muFc-muEndo-K1 comprises muFc-muEndo joinedby a polypeptide linker containing glycine and serine residues, to thefirst Kringle of murine angiostatin. The DNA construct was assembled asfollows.

[0094] The BamHI end of the HindIII-BamHI fragment encoding themuEndo-GlySer linker (Example 12) was ligated to the HindIII-XhoIfragment encoding the Kringle 1 of murine angiostatin (Example 10) viathe following adaptor: BamHI 5′ GA TCC TCA GGC C (SEQ ID NO:49)        G AGT CCG GTCGA (SEQ ID NO:50)                     HindIII

[0095] The adaptor has a HindIII′ sticky end, which upon ligation, wouldnot regenerate the HindIII site. Thus, the resulting HindIII-XhoIfragment, which encodes the muEndo-GlySer linker-Kringle 1, was ligatedto the pdCs-muFc(D₄K) vector for expression. High levels of expressionof muFc-muEndo-GlySer linker-K1 were obtained in both transient andstable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.

Example 14 Expression of muFc-muEndo-GlySer Linker-muAngio

[0096] The hybrid molecule muFc-muEndo-GlySer linker-muAngio comprisesmuFc-muEndo joined by a polypeptide linker containing glycine and serineresidues, to murine angiostatin. The DNA construct was assembledessentially as follows. The BamHI end of the HindIII-BamHI fragmentencoding the muEndo-GlySer linker (Example 12) was ligated to theHindIII-XhoI fragment encoding murine angiostatin via the adaptordescribed in Example 13. The resulting HindIII-XhoI fragment, whichencodes the muEndo-GlySer linker-muAngio, was ligated to thepdCs-muFc(D₄K) vector for expression. High levels of expression ofmuFc-muEndo-GlySer linker-muAngio were obtained in both transient andstable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.

Example 15 Expression of huAngio-huFc-huEndo

[0097] The hybrid molecule huAngio-huFc-huEndo comprises humanangiostatin joined by a peptide bond to huFc-huEndo. The DNA constructwas assembled as follows. A HindIII-XhoI fragment which encodes humanangiostatin without a STOP codon was first generated by PCR, so that thecodon for the last amino acid residue of angiostatin was followedimmediately by CTCGAG of the XhoI site. The HindIII at the 5′ end wasligated to an XbaI-AfIII fragment of the light chain signal peptide (Loet al., Protein Engineering (1998) 11:495) via a AfIII-HindIII′ adaptor:   AfIII 5′ TTA AGC GGC C (SEQ ID NO:51)         CG CGG GTCGA (SEQ IDNO:52)                 HindIII′

[0098] The HindIII′ sticky end of the adaptor, upon ligation, would notregenerate a HindIII site. At the 3′ end, the XhoI site was ligated tothe AfIII site of the AfIII-XhoI fragment encoding the huFc-hu-Endo viathe following XhoI′-AfIII adaptor:    XhoI′ 5′ TC GAC TCC GGC (SEQ IDNO:53)         G AGG CCG AATT (SEQ ID NO:54)                 AfIII

[0099] The XhoI sticky end of the adaptor, upon ligation, would notregenerate a XhoI site. The resulting XbaI-XhoI fragment encoding thesignal peptide-human angiostatin-huFc-human endostatin was cloned intothe pdCs vector for expression. High levels of expression of wereobtained in both transient and stable expression, as analyzed byanti-muFc ELISA and SDS-PAGE.

Example 16

[0100] Pharmacokinetics

[0101] In one set of pharmacokinetic studies, C57/BL6 mice withimplanted Lewis lung tumors at 100-200 mm³ were injected in the tailvein with 720 μg huFc-huAngio per mouse. The size of the tumors and thedosage of huFc-huAngio used in this study were chosen to simulate theactual treatment protocol described by O'Reilly (O'Reilly et al., (1996)Nature Medicine 2:689). Blood was harvested by retro-orbital bleeding at½, 1, 2, 4, 8, 24, and 48 hr. post injection. The blood samples wereanalyzed by anti-huFc ELISA followed by Western analysis. HuFc-huAngiowas found to have a circulating half-life of about 32 hr. in mouse andWestern analysis showed that over 90% of the hu-Fc-huAngio remained asan intact molecule in circulation.

[0102] The pharmacokinetic studies was also repeated in Swiss micewithout tumors at a dosage of 200 μg/mouse. In this case huFc-huAngiowas found to have a circulating half-life of about 33 hr.

[0103] Equivalents

[0104] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof Theforegoing embodiments are therefore to be considered in all respectsillustrative rather than limiting on the invention described herein.Scope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes which come within themeaning and range of equivalency of the claims are therefore intended tobe embraced therein.

1 54 1 696 DNA Homo sapiens CDS (1)..(696) Fc fragment of the humanimmunoglobulin gamma 1 gag ccc aaa tct tct gac aaa act cac aca tgc ccaccg tgc cca gca 48 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro ProCys Pro Ala 1 5 10 15 cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttcccc cca aaa ccc 96 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro 20 25 30 aag gac acc ctc atg atc tcc cgg acc cct gag gtc acatgc gtg gtg 144 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr CysVal Val 35 40 45 gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tggtac gtg 192 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp TyrVal 50 55 60 gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gagcag 240 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80 tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg caccag 288 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln85 90 95 gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc336 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100105 110 ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc384 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115120 125 cga gaa cca cag gtg tac acc ctg ccc cca tca cgg gag gag atg acc432 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130135 140 aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc480 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145150 155 160 gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aactac 528 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr165 170 175 aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctctat 576 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr180 185 190 agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtcttc 624 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe195 200 205 tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cagaag 672 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys210 215 220 agc ctc tcc ctg tcc ccg ggt aaa 696 Ser Leu Ser Leu Ser ProGly Lys 225 230 2 232 PRT Homo sapiens 2 Glu Pro Lys Ser Ser Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu AspPro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly LysGlu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 AspIle Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 3 549 DNA Homosapiens CDS (1)..(549) endostatin 3 cac agc cac cgc gac ttc cag ccg gtgctc cac ctg gtt gcg ctc aac 48 His Ser His Arg Asp Phe Gln Pro Val LeuHis Leu Val Ala Leu Asn 1 5 10 15 agc ccc ctg tca ggc ggc atg cgg ggcatc cgc ggg gcc gac ttc cag 96 Ser Pro Leu Ser Gly Gly Met Arg Gly IleArg Gly Ala Asp Phe Gln 20 25 30 tgc ttc cag cag gcg cgg gcc gtg ggg ctggcg ggc acc ttc cgc gcc 144 Cys Phe Gln Gln Ala Arg Ala Val Gly Leu AlaGly Thr Phe Arg Ala 35 40 45 ttc ctg tcc tcg cgc ctg cag gac ctg tac agcatc gtg cgc cgt gcc 192 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser IleVal Arg Arg Ala 50 55 60 gac cgc gca gcc gtg ccc atc gtc aac ctc aag gacgag ctg ctg ttt 240 Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp GluLeu Leu Phe 65 70 75 80 ccc agc tgg gag gct ctg ttc tca ggc tct gag ggtccg ctg aag ccc 288 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly ProLeu Lys Pro 85 90 95 ggg gca cgc atc ttc tcc ttt gac ggc aag gac gtc ctgagg cac ccc 336 Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu ArgHis Pro 100 105 110 acc tgg ccc cag aag agc gtg tgg cat ggc tcg gac cccaac ggg cgc 384 Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro AsnGly Arg 115 120 125 agg ctg acc gag agc tac tgt gag acg tgg cgg acg gaggct ccc tcg 432 Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu AlaPro Ser 130 135 140 gcc acg ggc cag gcc tcc tcg ctg ctg ggg ggc agg ctcctg ggg cag 480 Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu LeuGly Gln 145 150 155 160 agt gcc gcg agc tgc cat cac gcc tac atc gtg ctctgc att gag aac 528 Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu CysIle Glu Asn 165 170 175 agc ttc atg act gcc tcc aag 549 Ser Phe Met ThrAla Ser Lys 180 4 183 PRT Homo sapiens 4 His Ser His Arg Asp Phe Gln ProVal Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly MetArg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln Gln Ala Arg AlaVal Gly Leu Ala Gly Thr Phe Arg Ala 35 40 45 Phe Leu Ser Ser Arg Leu GlnAsp Leu Tyr Ser Ile Val Arg Arg Ala 50 55 60 Asp Arg Ala Ala Val Pro IleVal Asn Leu Lys Asp Glu Leu Leu Phe 65 70 75 80 Pro Ser Trp Glu Ala LeuPhe Ser Gly Ser Glu Gly Pro Leu Lys Pro 85 90 95 Gly Ala Arg Ile Phe SerPhe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 Thr Trp Pro Gln LysSer Val Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125 Arg Leu Thr GluSer Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser 130 135 140 Ala Thr GlyGln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln 145 150 155 160 SerAla Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn 165 170 175Ser Phe Met Thr Ala Ser Lys 180 5 30 DNA Artificial Sequence Descriptionof Artificial SequenceForward primer for human Fc-Endo 5 cc ccg ggt aaacac agc cac cgc gac ttc c 30 Pro Gly Lys His Ser His Arg Asp Phe 1 5 6 9PRT Artificial Sequence Description of Artificial SequenceForward primerfor human Fc-Endo 6 Pro Gly Lys His Ser His Arg Asp Phe 1 5 7 26 DNAArtificial Sequence Description of Artificial SequenceForward primer forhuman Fc-endo 7 c aag ctt cac agc cac cgc gac ttc c 26 Lys Leu His SerHis Arg Asp Phe 1 5 8 8 PRT Artificial Sequence Description ofArtificial SequenceForward primer for human Fc-endo 8 Lys Leu His SerHis Arg Asp Phe 1 5 9 26 DNA Artificial Sequence Description ofArtificial SequenceReverse primer for human Fc-Endo 9 cctcgagctacttggaggca gtcatg 26 10 1089 DNA Homo sapiens CDS (1)..(1089)angiostatin 10 aaa gtg tat ctc tca gag tgc aag act ggg aat gga aag aactac aga 48 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn TyrArg 1 5 10 15 ggg acg atg tcc aaa aca aaa aat ggc atc acc tgt caa aaatgg agt 96 Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys TrpSer 20 25 30 tcc act tct ccc cac aga cct aga ttc tca cct gct aca cac ccctca 144 Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser35 40 45 gag gga ctg gag gag aac tac tgc agg aat cca gac aac gat ccg cag192 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 5055 60 ggg ccc tgg tgc tat act act gat cca gaa aag aga tat gac tac tgc240 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 6570 75 80 gac att ctt gag tgt gaa gag gaa tgt atg cat tgc agt gga gaa aac288 Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn 8590 95 tat gac ggc aaa att tcc aag acc atg tct gga ctg gaa tgc cag gcc336 Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala 100105 110 tgg gac tct cag agc cca cac gct cat gga tac att cct tcc aaa ttt384 Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe 115120 125 cca aac aag aac ctg aag aag aat tac tgt cgt aac ccc gat agg gag432 Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu 130135 140 ctg cgg cct tgg tgt ttc acc acc gac ccc aac aag cgc tgg gaa ctt480 Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 145150 155 160 tgc gac atc ccc cgc tgc aca aca cct cca cca tct tct ggt cccacc 528 Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr165 170 175 tac cag tgt ctg aag gga aca ggt gaa aac tat cgc ggg aat gtggct 576 Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala180 185 190 gtt acc gtt tcc ggg cac acc tgt cag cac tgg agt gca cag acccct 624 Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro195 200 205 cac aca cat aac agg aca cca gaa aac ttc ccc tgc aaa aat ttggat 672 His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp210 215 220 gaa aac tac tgc cgc aat cct gac gga aaa agg gcc cca tgg tgccat 720 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His225 230 235 240 aca acc aac agc caa gtg cgg tgg gag tac tgt aag ata ccgtcc tgt 768 Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro SerCys 245 250 255 gac tcc tcc cca gta tcc acg gaa caa ttg gct ccc aca gcacca cct 816 Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala ProPro 260 265 270 gag cta acc cct gtg gtc cag gac tgc tac cat ggt gat ggacag agc 864 Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly GlnSer 275 280 285 tac cga ggc aca tcc tcc acc acc acc aca gga aag aag tgtcag tct 912 Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys GlnSer 290 295 300 tgg tca tct atg aca cca cac cgg cac cag aag acc cca gaaaac tac 960 Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu AsnTyr 305 310 315 320 cca aat gct ggc ctg aca atg aac tac tgc agg aat ccagat gcc gat 1008 Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro AspAla Asp 325 330 335 aaa ggc ccc tgg tgt ttt acc aca gac ccc agc gtc aggtgg gag tac 1056 Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg TrpGlu Tyr 340 345 350 tgc aac ctg aaa aaa tgc tca gga aca gaa gcg 1089 CysAsn Leu Lys Lys Cys Ser Gly Thr Glu Ala 355 360 11 363 PRT Homo sapiens11 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 1 510 15 Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser 2025 30 Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser 3540 45 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 5055 60 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 6570 75 80 Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn85 90 95 Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala100 105 110 Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser LysPhe 115 120 125 Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro AspArg Glu 130 135 140 Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys ArgTrp Glu Leu 145 150 155 160 Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro ProSer Ser Gly Pro Thr 165 170 175 Tyr Gln Cys Leu Lys Gly Thr Gly Glu AsnTyr Arg Gly Asn Val Ala 180 185 190 Val Thr Val Ser Gly His Thr Cys GlnHis Trp Ser Ala Gln Thr Pro 195 200 205 His Thr His Asn Arg Thr Pro GluAsn Phe Pro Cys Lys Asn Leu Asp 210 215 220 Glu Asn Tyr Cys Arg Asn ProAsp Gly Lys Arg Ala Pro Trp Cys His 225 230 235 240 Thr Thr Asn Ser GlnVal Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 245 250 255 Asp Ser Ser ProVal Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro 260 265 270 Glu Leu ThrPro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 275 280 285 Tyr ArgGly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 290 295 300 TrpSer Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr 305 310 315320 Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp 325330 335 Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr340 345 350 Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala 355 360 12 29DNA Artificial Sequence Description of Artificial SequenceForward primerfor human Fc-Angio 12 cc ccg ggt aag aaa gtg tat ctc tca gag 29 Pro GlyLys Lys Val Tyr Leu Ser Glu 1 5 13 9 PRT Artificial Sequence Descriptionof Artificial SequenceForward primer for human Fc-Angio 13 Pro Gly LysLys Val Tyr Leu Ser Glu 1 5 14 28 DNA Artificial Sequence Description ofArtificial SequenceForward primer for human Fc-Angio 14 c ccc aag cttaaa gtg tat ctc tca gag 28 Pro Lys Leu Lys Val Tyr Leu Ser Glu 1 5 15 9PRT Artificial Sequence Description of Artificial SequenceForward primerfor human Fc-Angio 15 Pro Lys Leu Lys Val Tyr Leu Ser Glu 1 5 16 30 DNAArtificial Sequence Description of Artificial SequenceReverse primer forhuman Fc-Angio 16 cccctcgagc tacgcttctg ttcctgagca 30 17 552 DNA Musmusculus CDS (1)..(552) endostatin 17 cat act cat cag gac ttt cag ccagtg ctc cac ctg gtg gca ctg aac 48 His Thr His Gln Asp Phe Gln Pro ValLeu His Leu Val Ala Leu Asn 1 5 10 15 acc ccc ctg tct gga ggc atg cgtggt atc cgt gga gca gat ttc cag 96 Thr Pro Leu Ser Gly Gly Met Arg GlyIle Arg Gly Ala Asp Phe Gln 20 25 30 tgc ttc cag caa gcc cga gcc gtg gggctg tcg ggc acc ttc cgg gct 144 Cys Phe Gln Gln Ala Arg Ala Val Gly LeuSer Gly Thr Phe Arg Ala 35 40 45 ttc ctg tcc tct agg ctg cag gat ctc tatagc atc gtg cgc cgt gct 192 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr SerIle Val Arg Arg Ala 50 55 60 gac cgg ggg tct gtg ccc atc gtc aac ctg aaggac gag gtg cta tct 240 Asp Arg Gly Ser Val Pro Ile Val Asn Leu Lys AspGlu Val Leu Ser 65 70 75 80 ccc agc tgg gac tcc ctg ttt tct ggc tcc cagggt caa gtg caa ccc 288 Pro Ser Trp Asp Ser Leu Phe Ser Gly Ser Gln GlyGln Val Gln Pro 85 90 95 ggg gcc cgc atc ttt tct ttt gac ggc aga gat gtcctg aga cac cca 336 Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val LeuArg His Pro 100 105 110 gcc tgg ccg cag aag agc gta tgg cac ggc tcg gacccc agt ggg cgg 384 Ala Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp ProSer Gly Arg 115 120 125 agg ctg atg gag agt tac tgt gag aca tgg cga actgaa act act ggg 432 Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr GluThr Thr Gly 130 135 140 gct aca ggt cag gcc tcc tcc ctg ctg tca ggc aggctc ctg gaa cag 480 Ala Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg LeuLeu Glu Gln 145 150 155 160 aaa gct gcg agc tgc cac aac agc tac atc gtcctg tgc att gag aat 528 Lys Ala Ala Ser Cys His Asn Ser Tyr Ile Val LeuCys Ile Glu Asn 165 170 175 agc ttc atg acc tct ttc tcc aaa 552 Ser PheMet Thr Ser Phe Ser Lys 180 18 184 PRT Mus musculus 18 His Thr His GlnAsp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro LeuSer Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe GlnGln Ala Arg Ala Val Gly Leu Ser Gly Thr Phe Arg Ala 35 40 45 Phe Leu SerSer Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala 50 55 60 Asp Arg GlySer Val Pro Ile Val Asn Leu Lys Asp Glu Val Leu Ser 65 70 75 80 Pro SerTrp Asp Ser Leu Phe Ser Gly Ser Gln Gly Gln Val Gln Pro 85 90 95 Gly AlaArg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Arg His Pro 100 105 110 AlaTrp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly 130 135140 Ala Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln 145150 155 160 Lys Ala Ala Ser Cys His Asn Ser Tyr Ile Val Leu Cys Ile GluAsn 165 170 175 Ser Phe Met Thr Ser Phe Ser Lys 180 19 699 DNA Musmusculus CDS (1)..(699) Fc 19 gag ccc aga ggg ccc aca atc aag ccc tgtcct cca tgc aaa tgc cca 48 Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys ProPro Cys Lys Cys Pro 1 5 10 15 gca cct aac ctc ttg ggt gga cca tcc gtcttc atc ttc cct cca aag 96 Ala Pro Asn Leu Leu Gly Gly Pro Ser Val PheIle Phe Pro Pro Lys 20 25 30 atc aag gat gta ctc atg atc tcc ctg agc cccata gtc aca tgt gtg 144 Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro IleVal Thr Cys Val 35 40 45 gtg gtg gat gtg agc gag gat gac cca gat gtc cagatc agc tgg ttt 192 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln IleSer Trp Phe 50 55 60 gtg aac aac gtg gaa gta cac aca gct cag aca caa acccat aga gag 240 Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr HisArg Glu 65 70 75 80 gat tac aac agt act ctc cgg gtg gtc agt gcc ctc cccatc cag cac 288 Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro IleGln His 85 90 95 cag gac tgg atg agt ggc aag gag ttc aaa tgc aag gtc aacaac aaa 336 Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn AsnLys 100 105 110 gac ctc cca gcg ccc atc gag aga acc atc tca aaa ccc aaaggg tca 384 Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys GlySer 115 120 125 gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaagag atg 432 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu GluMet 130 135 140 act aag aaa cag gtc act ctg acc tgc atg gtc aca gac ttcatg cct 480 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe MetPro 145 150 155 160 gaa gac att tac gtg gag tgg acc aac aac ggg aaa acagag cta aac 528 Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr GluLeu Asn 165 170 175 tac aag aac act gaa cca gtc ctg gac tct gat ggt tcttac ttc atg 576 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser TyrPhe Met 180 185 190 tac agc aag ctg aga gtg gaa aag aag aac tgg gtg gaaaga aat agc 624 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu ArgAsn Ser 195 200 205 tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat caccac acg act 672 Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His HisThr Thr 210 215 220 aag agc ttc tcc cgg acc ccg ggt aaa 699 Lys Ser PheSer Arg Thr Pro Gly Lys 225 230 20 233 PRT Mus musculus 20 Glu Pro ArgGly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro 1 5 10 15 Ala ProAsn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 20 25 30 Ile LysAsp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val 35 40 45 Val ValAsp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe 50 55 60 Val AsnAsn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu 65 70 75 80 AspTyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His 85 90 95 GlnAsp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105 110Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser 115 120125 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130135 140 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro145 150 155 160 Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr GluLeu Asn 165 170 175 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly SerTyr Phe Met 180 185 190 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp ValGlu Arg Asn Ser 195 200 205 Tyr Ser Cys Ser Val Val His Glu Gly Leu HisAsn His His Thr Thr 210 215 220 Lys Ser Phe Ser Arg Thr Pro Gly Lys 225230 21 29 DNA Artificial Sequence Description of ArtificialSequenceForward primer for mouse Fc-Endo 21 c ccc aag ctt cat act catcag gac ttt c 29 Pro Lys Leu His Thr His Gln Asp Phe 1 5 22 9 PRTArtificial Sequence Description of Artificial SequenceForward primer formouse Fc-Endo 22 Pro Lys Leu His Thr His Gln Asp Phe 1 5 23 28 DNAArtificial Sequence Description of Artificial SequenceReverse primer formouse Fc-Endo 23 cccctcgagc tatttggaga aagaggtc 28 24 1086 DNA Musmusculus CDS (1)..(1086) Angiostatin 24 gtg tat ctg tca gaa tgt aag accggc atc ggc aac ggc tac aga gga 48 Val Tyr Leu Ser Glu Cys Lys Thr GlyIle Gly Asn Gly Tyr Arg Gly 1 5 10 15 acc atg tcc agg aca aag agt ggtgtt gcc tgt caa aag tgg ggt gcc 96 Thr Met Ser Arg Thr Lys Ser Gly ValAla Cys Gln Lys Trp Gly Ala 20 25 30 acg ttc ccc cac gta ccc aac tac tctccc agt aca cat ccc aat gag 144 Thr Phe Pro His Val Pro Asn Tyr Ser ProSer Thr His Pro Asn Glu 35 40 45 gga cta gaa gag aac tac tgt agg aac ccagac aat gat gaa caa ggg 192 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro AspAsn Asp Glu Gln Gly 50 55 60 cct tgg tgc tac act aca gat ccg gac aag agatat gac tac tgc aac 240 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg TyrAsp Tyr Cys Asn 65 70 75 80 att cct gaa tgt gaa gag gaa tgc atg tac tgcagt gga gaa aag tat 288 Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys SerGly Glu Lys Tyr 85 90 95 gag ggc aaa atc tcc aag acc atg tct gga ctt gactgc cag gcc tgg 336 Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Asp CysGln Ala Trp 100 105 110 gat tct cag agc cca cat gct cat gga tac atc cctgcc aaa ttt cca 384 Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro AlaLys Phe Pro 115 120 125 agc aag aac ctg aag atg aat tat tgc cac aac cctgac ggg gag cca 432 Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro AspGly Glu Pro 130 135 140 agg ccc tgg tgc ttc aca aca gac ccc acc aaa cgctgg gaa tac tgt 480 Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg TrpGlu Tyr Cys 145 150 155 160 gac atc ccc cgc tgc aca aca ccc ccg ccc ccaccc agc cca acc tac 528 Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro ProSer Pro Thr Tyr 165 170 175 caa tgt ctg aaa gga aga ggt gaa aat tac cgaggg acc gtg tct gtc 576 Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg GlyThr Val Ser Val 180 185 190 acc gtg tct ggg aaa acc tgt cag cgc tgg agtgag caa acc cct cat 624 Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser GluGln Thr Pro His 195 200 205 agg cac aac agg aca cca gaa aat ttc ccc tgcaaa aat ctg gaa gag 672 Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys LysAsn Leu Glu Glu 210 215 220 aac tac tgc cgg aac cca gat gga gaa act gctccc tgg tgc tat acc 720 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala ProTrp Cys Tyr Thr 225 230 235 240 act gac agc cag ctg agg tgg gag tac tgtgag att cca tcc tgc gag 768 Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys GluIle Pro Ser Cys Glu 245 250 255 tcc tca gca tca cca gac cag tca gat tcctca gtt cca cca gag gag 816 Ser Ser Ala Ser Pro Asp Gln Ser Asp Ser SerVal Pro Pro Glu Glu 260 265 270 caa aca cct gtg gtc cag gaa tgc tac cagagc gat ggg cag agc tat 864 Gln Thr Pro Val Val Gln Glu Cys Tyr Gln SerAsp Gly Gln Ser Tyr 275 280 285 cgg ggt aca tcg tcc act acc atc aca gggaag aag tgc cag tcc tgg 912 Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly LysLys Cys Gln Ser Trp 290 295 300 gca gct atg ttt cca cac agg cat tcg aagacc cca gag aac ttc cca 960 Ala Ala Met Phe Pro His Arg His Ser Lys ThrPro Glu Asn Phe Pro 305 310 315 320 gat gct ggc ttg gag atg aac tac tgcagg aac ccg gat ggt gac aag 1008 Asp Ala Gly Leu Glu Met Asn Tyr Cys ArgAsn Pro Asp Gly Asp Lys 325 330 335 ggc cct tgg tgc tac acc act gac ccgagc gtc agg tgg gaa tac tgc 1056 Gly Pro Trp Cys Tyr Thr Thr Asp Pro SerVal Arg Trp Glu Tyr Cys 340 345 350 aac ctg aag cgg tgc tca gag aca ggaggg 1086 Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly 355 360 25 362 PRT Musmusculus 25 Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr ArgGly 1 5 10 15 Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys TrpGly Ala 20 25 30 Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His ProAsn Glu 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp GluGln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp TyrCys Asn 65 70 75 80 Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser GlyGlu Lys Tyr 85 90 95 Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Asp CysGln Ala Trp 100 105 110 Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile ProAla Lys Phe Pro 115 120 125 Ser Lys Asn Leu Lys Met Asn Tyr Cys His AsnPro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro ThrLys Arg Trp Glu Tyr Cys 145 150 155 160 Asp Ile Pro Arg Cys Thr Thr ProPro Pro Pro Pro Ser Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg GlyGlu Asn Tyr Arg Gly Thr Val Ser Val 180 185 190 Thr Val Ser Gly Lys ThrCys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 Arg His Asn Arg ThrPro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys ArgAsn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr 225 230 235 240 Thr AspSer Gln Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu 245 250 255 SerSer Ala Ser Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu 260 265 270Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr 275 280285 Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp 290295 300 Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro305 310 315 320 Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp GlyAsp Lys 325 330 335 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg TrpGlu Tyr Cys 340 345 350 Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly 355 36026 31 DNA Artificial Sequence Description of Artificial SequenceForwardprimer for mouse Fc-Angio 26 c ccc aag ctt gtg tat ctg tca gaa tgt aag31 Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys 1 5 10 27 10 PRT ArtificialSequence Description of Artificial SequenceForward primer for mouseFc-Angio 27 Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys 1 5 10 28 30 DNAArtificial Sequence Description of Artificial SequenceReverse primer formouse Fc-Angio 28 cccctcgagc taccctcctg tctctgagca 30 29 29 DNAArtificial Sequence Description of Artificial SequenceForward primer forcanine Fc 29 cc tta agc gaa aat gga aga gtt cct cgc 29 Leu Ser Glu AsnGly Arg Val Pro Arg 1 5 30 9 PRT Artificial Sequence Description ofArtificial SequenceForward primer for canine Fc 30 Leu Ser Glu Asn GlyArg Val Pro Arg 1 5 31 40 DNA Artificial Sequence Description ofArtificial SequenceReverse primer for canine Fc 31 cctcgagtca tttacccggggaatgggaga gggatttctg 40 32 702 DNA Canis familiaris CDS (1)..(702) Fc32 gaa aat gga aga gtt cct cgc cca cct gat tgt ccc aaa tgc cca gcc 48Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala 1 5 1015 cct gaa atg ctg gga ggg cct tcg gtc ttc atc ttt ccc ccg aaa ccc 96Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 20 25 30aag gac acc ctc ttg att gcc cga aca cct gag gtc aca tgt gtg gtg 144 LysAsp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 gtggat ctg gga cca gaa gac cct gag gtg cag atc agc tgg ttc gtg 192 Val AspLeu Gly Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val 50 55 60 gac ggtaag cag atg caa aca gcc aag act cag cct cgt gag gag cag 240 Asp Gly LysGln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln 65 70 75 80 ttc aatggc acc tac cgt gtg gtc agt gtc ctc ccc att ggg cac cag 288 Phe Asn GlyThr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln 85 90 95 gac tgg ctcaag ggg aag cag ttc acg tgc aaa gtc aac aac aaa gcc 336 Asp Trp Leu LysGly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala 100 105 110 ctc cca tccccg atc gag agg acc atc tcc aag gcc aga ggg cag gcc 384 Leu Pro Ser ProIle Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 115 120 125 cat cag cccagt gtg tat gtc ctg ccg cca tcc cgg gag gag ttg agc 432 His Gln Pro SerVal Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 130 135 140 aag aac acagtc agc ttg aca tgc ctg atc aaa gac ttc ttc cca cct 480 Lys Asn Thr ValSer Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 145 150 155 160 gac attgat gtg gag tgg cag agc aat gga cag cag gag cct gag agc 528 Asp Ile AspVal Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 165 170 175 aag taccgc acg acc ccg ccc cag ctg gac gag gac ggg tcc tac ttc 576 Lys Tyr ArgThr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 180 185 190 ctg tacagc aag ctc tct gtg gac aag agc cgc tgg cag cgg gga gac 624 Leu Tyr SerLys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 195 200 205 acc ttcata tgt gcg gtg atg cat gaa gct cta cac aac cac tac aca 672 Thr Phe IleCys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 cag aaatcc ctc tcc cat tct ccg ggt aaa 702 Gln Lys Ser Leu Ser His Ser Pro GlyLys 225 230 33 234 PRT Canis familiaris 33 Glu Asn Gly Arg Val Pro ArgPro Pro Asp Cys Pro Lys Cys Pro Ala 1 5 10 15 Pro Glu Met Leu Gly GlyPro Ser Val Phe Ile Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Leu IleAla Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Leu Gly Pro GluAsp Pro Glu Val Gln Ile Ser Trp Phe Val 50 55 60 Asp Gly Lys Gln Met GlnThr Ala Lys Thr Gln Pro Arg Glu Glu Gln 65 70 75 80 Phe Asn Gly Thr TyrArg Val Val Ser Val Leu Pro Ile Gly His Gln 85 90 95 Asp Trp Leu Lys GlyLys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala 100 105 110 Leu Pro Ser ProIle Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 115 120 125 His Gln ProSer Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 130 135 140 Lys AsnThr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro 145 150 155 160Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 165 170175 Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 180185 190 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp195 200 205 Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His TyrThr 210 215 220 Gln Lys Ser Leu Ser His Ser Pro Gly Lys 225 230 34 552DNA Canis familiaris CDS (1)..(552) Endostatin 34 cac acc cac cag gacttc cag ccg gtg ctg cac ctg gtg gcc ctg aac 48 His Thr His Gln Asp PheGln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 agc ccg cag ccg ggcggc atg cga ggc atc cgg gga gcg gac ttc cag 96 Ser Pro Gln Pro Gly GlyMet Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 tgc ttc cag cag gcg cgcgcc gcg ggg ctg gcc ggc acc ttc cgg gcc 144 Cys Phe Gln Gln Ala Arg AlaAla Gly Leu Ala Gly Thr Phe Arg Ala 35 40 45 ttc ctg tcg tcg cgg ctg caggac ctc tac agc atc gtg cgc cgc gcc 192 Phe Leu Ser Ser Arg Leu Gln AspLeu Tyr Ser Ile Val Arg Arg Ala 50 55 60 gac cgc acc ggg gtg ccc gtc gtcaac ctc agg gac gag gtg ctc ttc 240 Asp Arg Thr Gly Val Pro Val Val AsnLeu Arg Asp Glu Val Leu Phe 65 70 75 80 ccc agc tgg gag gcc tta ttc tcgggc tcc gag ggc cag ctg aag ccc 288 Pro Ser Trp Glu Ala Leu Phe Ser GlySer Glu Gly Gln Leu Lys Pro 85 90 95 ggg gcc cgc atc ttc tct ttc gac ggcaga gat gtc ctg cag cac ccc 336 Gly Ala Arg Ile Phe Ser Phe Asp Gly ArgAsp Val Leu Gln His Pro 100 105 110 gcc tgg ccc cgg aag agc gtg tgg cacggc tcc gac ccc agc ggg cgc 384 Ala Trp Pro Arg Lys Ser Val Trp His GlySer Asp Pro Ser Gly Arg 115 120 125 cgc ctg acc gac agc tac tgc gag acgtgg cgg acg gag gcc ccg gcg 432 Arg Leu Thr Asp Ser Tyr Cys Glu Thr TrpArg Thr Glu Ala Pro Ala 130 135 140 gcc acc ggg cag gcg tcg tcg ctg ctggcg ggc agg ctg ctg gag cag 480 Ala Thr Gly Gln Ala Ser Ser Leu Leu AlaGly Arg Leu Leu Glu Gln 145 150 155 160 gag gcc gcg agc tgc cgc cac gccttc gtg gtg ctc tgc atc gag aac 528 Glu Ala Ala Ser Cys Arg His Ala PheVal Val Leu Cys Ile Glu Asn 165 170 175 agc gtc atg acc tcc ttc tcc aag552 Ser Val Met Thr Ser Phe Ser Lys 180 35 184 PRT Canis familiaris 35His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 1015 Ser Pro Gln Pro Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 2530 Cys Phe Gln Gln Ala Arg Ala Ala Gly Leu Ala Gly Thr Phe Arg Ala 35 4045 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala 50 5560 Asp Arg Thr Gly Val Pro Val Val Asn Leu Arg Asp Glu Val Leu Phe 65 7075 80 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Gln Leu Lys Pro 8590 95 Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Gln His Pro100 105 110 Ala Trp Pro Arg Lys Ser Val Trp His Gly Ser Asp Pro Ser GlyArg 115 120 125 Arg Leu Thr Asp Ser Tyr Cys Glu Thr Trp Arg Thr Glu AlaPro Ala 130 135 140 Ala Thr Gly Gln Ala Ser Ser Leu Leu Ala Gly Arg LeuLeu Glu Gln 145 150 155 160 Glu Ala Ala Ser Cys Arg His Ala Phe Val ValLeu Cys Ile Glu Asn 165 170 175 Ser Val Met Thr Ser Phe Ser Lys 180 3641 DNA Artificial Sequence Description of Artificial SequenceHindIII/DraIII linker top strand 36 ag ctt cac acc cac cag gac ttc cagccg gtg ctg cac ctg 41 Leu His Thr His Gln Asp Phe Gln Pro Val Leu HisLeu 1 5 10 37 13 PRT Artificial Sequence Description of ArtificialSequence HindIII/DraIII linker top strand 37 Leu His Thr His Gln Asp PheGln Pro Val Leu His Leu 1 5 10 38 34 DNA Artificial Sequence Descriptionof Artificial Sequence HindIII/DraIII linker bottom strand 38 gtgcagcaccggctggaagt cctggtgggt gtga 34 39 1077 DNA Canis familiaris CDS(1)..(1077) angiostatin 39 ata tat ctt tca gag tgc aag act gga aat gggaaa acc tac agg ggg 48 Ile Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly LysThr Tyr Arg Gly 1 5 10 15 acc atg gcc aaa acg aag aat gat gtt gcc tgtcaa aaa tgg agt gac 96 Thr Met Ala Lys Thr Lys Asn Asp Val Ala Cys GlnLys Trp Ser Asp 20 25 30 aat tct ccg cac aaa cct aac tat acg cct gag aagcac ccc ttg gag 144 Asn Ser Pro His Lys Pro Asn Tyr Thr Pro Glu Lys HisPro Leu Glu 35 40 45 ggg ctg gag gag aac tat tgc agg aac cct gac aac gacgag aac ggg 192 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp GluAsn Gly 50 55 60 ccc tgg tgc tac acc aca aac cca gac gtg agg ttc gac tactgc aac 240 Pro Trp Cys Tyr Thr Thr Asn Pro Asp Val Arg Phe Asp Tyr CysAsn 65 70 75 80 att cca gaa tgt gaa gag gaa tgt atg cat tgc agt ggg gaaaat tat 288 Ile Pro Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu AsnTyr 85 90 95 gag ggc aaa att tcc aag aca aag tct gga ctc gag tgc caa gcctgg 336 Glu Gly Lys Ile Ser Lys Thr Lys Ser Gly Leu Glu Cys Gln Ala Trp100 105 110 aac tct caa acc cca cat gct cat gga tat att cct tcc aaa tttcca 384 Asn Ser Gln Thr Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro115 120 125 agc aag aac ttg aag atg aat tac tgc cgt aac cct gat ggg gagccc 432 Ser Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro130 135 140 cgc cca tgg tgt ttc acc atg gat ccc aac aaa cgc tgg gaa ttctgt 480 Arg Pro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys145 150 155 160 gac att ccc cgc tgt aca aca cca cca ccc cct tcg ggc ccaacg tac 528 Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Ser Gly Pro ThrTyr 165 170 175 cag tgt ctg aag ggc aga ggg gag agc tac cga ggg aag gtgtcc gtc 576 Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val SerVal 180 185 190 act gtc tct gga cat aca tgt cag cac tgg agt gaa cag acccct cac 624 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Glu Gln Thr ProHis 195 200 205 aag cac aac agg acc cca gaa aac ttc cct tgc aaa aat ttggat gaa 672 Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu AspGlu 210 215 220 aac tac tgt cgc aac cct gat gga gaa aca gct cca tgg tgctac aca 720 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys TyrThr 225 230 235 240 acc aac agt gag gtg agg tgg gaa cac tgc cag att ccgtcc tgt gag 768 Thr Asn Ser Glu Val Arg Trp Glu His Cys Gln Ile Pro SerCys Glu 245 250 255 tcc tct cca ata acc aca gaa tat ttg gat gcc cca gcttca gtg cca 816 Ser Ser Pro Ile Thr Thr Glu Tyr Leu Asp Ala Pro Ala SerVal Pro 260 265 270 cct gaa caa act cct gtg gtc cag gag tgc tac cac ggcaat ggg cag 864 Pro Glu Gln Thr Pro Val Val Gln Glu Cys Tyr His Gly AsnGly Gln 275 280 285 agt tat cga ggc aca tca tcc act act atc aca gga agaaaa tgt cag 912 Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg LysCys Gln 290 295 300 tct tgg tca tct atg aca cca cac cga cat gag aag acccca gaa cac 960 Ser Trp Ser Ser Met Thr Pro His Arg His Glu Lys Thr ProGlu His 305 310 315 320 ttc ccg gag gct ggc ctg aca atg aac tac tgc aggaat ccc gac gcc 1008 Phe Pro Glu Ala Gly Leu Thr Met Asn Tyr Cys Arg AsnPro Asp Ala 325 330 335 gac aaa agc cct tgg tgt tac acc acc gac ccc tctgtg cgc tgg gag 1056 Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Ser ValArg Trp Glu 340 345 350 ttc tgt aac ttg aga aaa tgc 1077 Phe Cys Asn LeuArg Lys Cys 355 40 359 PRT Canis familiaris 40 Ile Tyr Leu Ser Glu CysLys Thr Gly Asn Gly Lys Thr Tyr Arg Gly 1 5 10 15 Thr Met Ala Lys ThrLys Asn Asp Val Ala Cys Gln Lys Trp Ser Asp 20 25 30 Asn Ser Pro His LysPro Asn Tyr Thr Pro Glu Lys His Pro Leu Glu 35 40 45 Gly Leu Glu Glu AsnTyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly 50 55 60 Pro Trp Cys Tyr ThrThr Asn Pro Asp Val Arg Phe Asp Tyr Cys Asn 65 70 75 80 Ile Pro Glu CysGlu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 Glu Gly Lys IleSer Lys Thr Lys Ser Gly Leu Glu Cys Gln Ala Trp 100 105 110 Asn Ser GlnThr Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro 115 120 125 Ser LysAsn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 ArgPro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys 145 150 155160 Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Ser Gly Pro Thr Tyr 165170 175 Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val Ser Val180 185 190 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Glu Gln Thr ProHis 195 200 205 Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn LeuAsp Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro TrpCys Tyr Thr 225 230 235 240 Thr Asn Ser Glu Val Arg Trp Glu His Cys GlnIle Pro Ser Cys Glu 245 250 255 Ser Ser Pro Ile Thr Thr Glu Tyr Leu AspAla Pro Ala Ser Val Pro 260 265 270 Pro Glu Gln Thr Pro Val Val Gln GluCys Tyr His Gly Asn Gly Gln 275 280 285 Ser Tyr Arg Gly Thr Ser Ser ThrThr Ile Thr Gly Arg Lys Cys Gln 290 295 300 Ser Trp Ser Ser Met Thr ProHis Arg His Glu Lys Thr Pro Glu His 305 310 315 320 Phe Pro Glu Ala GlyLeu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala 325 330 335 Asp Lys Ser ProTrp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu 340 345 350 Phe Cys AsnLeu Arg Lys Cys 355 41 12 DNA Artificial Sequence Description ofArtificial Sequencepalindromic linker where the STOP codon TGA isfollowed by an XhoI site 41 tgactcgagt ca 12 42 21 DNA ArtificialSequence Description of Artificial SequenceMutagenic primer for murineangiostatin 42 ggg cct tgg agc tac act aca 21 Gly Pro Trp Ser Tyr ThrThr 1 5 43 7 PRT Artificial Sequence Description of ArtificialSequenceMutagenic primer for murine angiostatin 43 Gly Pro Trp Ser TyrThr Thr 1 5 44 34 DNA Artificial Sequence Description of ArtificialSequenceprimer used to introduce HindIII into murine angiostatin 44gcggatcc aag ctt agt aca cat ccc aat gag gg 34 Lys Leu Ser Thr His ProAsn Glu 1 5 45 8 PRT Artificial Sequence Description of ArtificialSequenceprimer used to introduce HindIII into murine angiostatin 45 LysLeu Ser Thr His Pro Asn Glu 1 5 46 59 DNA Artificial SequenceDescription of Artificial SequenceBspHI/BamHI linker top strand 46 c atgacc tct ttc tcc aaa tcg agc ggg ggc agc ggg ggc gga ggc agc 49 Met ThrSer Phe Ser Lys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 ggcggg ggc g 59 Gly Gly Gly 47 19 PRT Artificial Sequence Description ofArtificial SequenceBspHI/BamHI linker top strand 47 Met Thr Ser Phe SerLys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly 48 59DNA Artificial Sequence Description of Artificial SequenceBspHI/BamHIlinker bottom strand 48 gatccgcccc cgccgctgcc tccgcccccg ctgcccccgctcgatttgga gaaagaggt 59 49 12 DNA Artificial Sequence Description ofArtificial Sequence BamHI/HindIII linker top strand 49 ga tcc tca ggc c12 Ser Ser Gly 1 50 12 DNA Artificial Sequence Description of ArtificialSequence BamHI/HindIII linker bottom strand 50 agctggcctg ag 12 51 10DNA Artificial Sequence Description of Artificial Sequence AflII/HindIIIlinker top strand 51 tta agc ggc c 10 Leu Ser Gly 1 52 10 DNA ArtificialSequence Description of Artificial Sequence AflII/HindIII linker bottomstrand 52 agctgggcgc 10 53 11 DNA Artificial Sequence Description ofArtificial SequenceXhoI/AflII linker top strand 53 tc gac tcc ggc 11 AspSer Gly 1 54 11 DNA Artificial Sequence Description of ArtificialSequenceXhoI/AflII linker bottom strand 54 ttaagccgga g 11

What is claimed is:
 1. A DNA molecule encoding a fusion proteincomprising: (a) a signal sequence; (b) an immunoglobulin Fe region; and(c) a target protein sequence selected from the group consisting ofangiostatin, endostatin, a plasminogen fragment having angiostatinactivity, a collagen XVIII fragment having endostatin activity, andcombinations thereof.
 2. The DNA of claim 1 wherein said signalsequence, said immunoglobulin Fc region and said target protein sequenceare encoded serially in a 5′ to 3′ direction.
 3. The DNA of claim 1,wherein said signal sequence, said target sequence, and saidimmunoglobulin Fc region are encoded serially in a 5′ to 3′ direction.4. The DNA of claim 1 wherein said immunoglobulin Fc region comprises animmunoglobulin hinge region.
 5. The DNA of claim 1 wherein saidimmunoglobulin Fc region comprises an immunoglobulin hinge region and animmunoglobulin constant heavy chain domain.
 6. The DNA of claim 1wherein said immunoglobulin Fc region comprises a hinge region and anCH₃ domain.
 7. The DNA of claim 1 wherein said immunoglobulin Fc regionlacks at least the CH₁ domain.
 8. The DNA of claim 1 wherein saidimmunoglobulin Fc region encodes at least a portion of immunoglobulingamma.
 9. A replicable expression vector for transfecting a mammaliancell, said vector comprising the DNA of claim
 1. 10. A mammalian cellharboring the DNA of claim
 1. 11. A fusion protein comprising animmunoglobulin Fc region, and a target protein selected from the groupconsisting of angiostatin, endostatin, a plasminogen fragment havingangiostatin activity, a collagen XVIII fragment having endostatinactivity, and combinations thereof.
 12. The fusion protein of claim 11wherein said plasminogen fragment has molecular weight of approximately40 kD and comprises an amino acid sequence set forth in SEQ ID No: 3.13. The fusion protein of claim 11 wherein said target protein comprisesamino acid sequence set forth in SEQ ID No:
 3. 14. The fusion protein ofclaim 11 wherein of said collagen XVIII fragment comprises the aminoacid sequence set forth in SEQ ID No:
 1. 15. The fusion protein of claim11 wherein said target protein comprises at least two molecules selectedfrom the group consisting of angiostatin, endostatin, a plasminogenfragment, and a collagen XVIII fragment, wherein said two molecules arelinked by a polypeptide linker.
 16. The fusion protein of claim 11wherein said target protein is linked to an N-terminal end of saidimmunoglobulin Fc region.
 17. The fusion protein of claim 11 whereinsaid target protein is linked to a C-terminal end of said immunoglobulinFc region.
 18. A multimeric protein comprising at least two fusionproteins of claim 11 linked via a disulfide bond.
 19. The multimericprotein of claim 18 wherein the target protein of at least one saidfusion protein is angiostatin and the target protein of at least onesaid fusion protein is endostatin.
 20. The multimeric protein of claim18 wherein the target protein of both of said fusion proteins isangiostatin.
 21. The multimeric protein of claim 18 wherein the targetprotein of both of said fusion proteins is endostatin.
 22. The fusionprotein of claim 11 further comprising a second target protein selectedfrom the group consisting of angiostatin, endostatin, a plasminogenfragment having angiostatin activity, and a collagen XVIII fragmenthaving endostatin activity.
 23. The fusion protein of claim 22 whereinsaid second target protein is linked by a polypeptide linker to saidfirst target protein.
 24. The fusion protein of claim 22 wherein saidfirst target protein is connected to an N-terminal end of saidimmunoglobulin Fc region and said second target protein is connected toa C-terminal end of said immunoglobulin Fc region.
 25. A multimericfusion protein comprising at least two fusion proteins of claim 11,wherein said fusion proteins are linked by a polypeptide bond.
 26. Amethod of producing a fusion protein, the method comprising the stepsof: a) providing the mammalian cell of claim 10; and b) culturing themammalian cell to produce said fusion protein.
 27. The method of claim26 comprising the additional step of collecting said fusion protein. 28.The method of claim 26 comprising the additional step of cleaving saidimmunoglobulin Fc region from said target protein.
 29. A method oftreating a condition mediated by angiogenesis comprising the step ofadministering the DNA of claim 1 to a mammal in need of an angiogenesisinhibitor.
 30. A method of treating a condition mediated by angiogenesiscomprising the step of administering the vector of claim 9 to a mammalin need of an angiogenesis inhibitor.
 31. A method of treating acondition alleviated by the administration of angiostatin or endostatincomprising the step of administering an effective amount of the fusionprotein of claim 11 to a mammal having said condition.